Crispr/cas dropout screening platform to reveal genetic vulnerabilities associated with tau aggregation

ABSTRACT

Cas-protein-ready tau biosensor cells, CRISPR/Cas synergistic activation mediator (SAM)-ready tau biosensor cells, and methods of making and using such cells to screen for genetic vulnerability associated with tau aggregation are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/821,384, filed Mar. 17, 2020, which claims the benefit of U.S.Application No. 62/820,101, filed Mar. 18, 2019, which is hereinincorporated by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS AN XML FILE VIA EFS WEB

The Sequence Listing written in file 599172SEQLIST.xml is 85.4kilobytes, was created on Aug. 30, 2023, and is hereby incorporated byreference.

BACKGROUND

Abnormal aggregation or fibrillization of proteins is a defining featureof many diseases, notably including a number of neurodegenerativediseases such as Alzheimer's disease (AD), Parkinson's disease (PD),frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS),chronic traumatic encephalopathy (CTE), Creutzfeldt-Jakob disease (CJD),and others. In many of these diseases, the fibrillization of certainproteins into insoluble aggregates is not only a hallmark of disease,but has also been implicated as a causative factor of neurotoxicity.Furthermore, these diseases are characterized by propagation ofaggregate pathology through the central nervous system followingstereotypical patterns, a process which correlates with diseaseprogression. The identification of genes and genetic pathways thatmodify the processes of abnormal protein aggregation, or cell-to-cellpropagation of aggregates, are therefore of great value in betterunderstanding neurodegenerative disease etiology as well as in devisingstrategies for therapeutic intervention.

SUMMARY

Provided herein are methods of screening for genetic vulnerabilitiesassociated with tau aggregation. Also provided herein are Cas-taubiosensor cells or populations of such cells and in vitro cultures ofCas-tau biosensor cells and a culture medium. Also provided herein areCRISPR/Cas synergistic activation mediator (SAM)-tau biosensor cells orpopulations of such cells and in vitro cultures of SAM-tau biosensorcells and a culture medium.

In one aspect, provided are methods of screening for geneticvulnerabilities associated with tau aggregation. Some such methodscomprise: (a) providing an aggregation-positive population of cells andan aggregation-negative population of cells, wherein each population ofcells comprises a Cas protein, a first tau repeat domain linked to afirst reporter, and a second tau repeat domain linked to a secondreporter, wherein in the aggregation-positive population of cells thefirst tau repeat domain linked to the first reporter and the second taurepeat domain linked to the second reporter stably present in anaggregated state, and wherein in the aggregation-negative population ofcells the first tau repeat domain linked to the first reporter and thesecond tau repeat domain linked to the second reporter do not stablypresent in an aggregated state; (b) introducing into each population ofcells a library comprising a plurality of unique guide RNAs that targeta plurality of genes, wherein the plurality of unique guide RNAs formcomplexes with the Cas protein, and the Cas protein cleaves theplurality of genes resulting in knockout of gene function; and (c)determining abundance of each of the plurality of unique guide RNAs at aplurality of time points over a time course in each population of cells,wherein depletion of a guide RNA in the aggregation-positive populationof cells but not in the aggregation-negative population of cells or amore dramatic depletion pattern of a guide RNA over the time course inthe aggregation-positive population of cells relative to theaggregation-negative population of cells indicates that the genetargeted by the guide RNA exhibits synthetic lethality with tau proteinaggregates and is a genetic vulnerability associated with tauaggregation or is a candidate genetic vulnerability associated tauaggregation (e.g., for further testing via secondary screens). GuideRNAs targeting such genes cause selective cell death in the Agg[+] cellsbut not in the Agg[−] cells.

In some such methods, the first tau repeat domain and/or the second taurepeat domain is a human tau repeat domain. In some such methods, thefirst tau repeat domain and/or the second tau repeat domain comprises apro-aggregation mutation. Optionally, the first tau repeat domain and/orthe second tau repeat domain comprises a tau P301S mutation. In somesuch methods, the first tau repeat domain and/or the second tau repeatdomain comprises a tau four-repeat domain. In some such methods, thefirst tau repeat domain and/or the second tau repeat domain comprisesSEQ ID NO: 11. In some such methods, the first tau repeat domain and thesecond tau repeat domain are the same. In some such methods, the firsttau repeat domain and the second tau repeat domain are the same and eachcomprises tau four-repeat domain comprising a tau P301S mutation.

In some such methods, the first reporter and the second reporter arefluorescent proteins. Optionally, the first reporter and the secondreporter are a fluorescence resonance energy transfer (FRET) pair.Optionally, the first reporter is cyan fluorescent protein (CFP) and thesecond reporter is yellow fluorescent protein (YFP).

In some such methods, the Cas protein is a Cas9 protein. Optionally, theCas protein is Streptococcus pyogenes Cas9. Optionally, the Cas proteincomprises SEQ ID NO: 21. Optionally, the Cas protein is encoded by acoding sequence comprising the sequence set forth in SEQ ID NO: 22.

In some such methods, the Cas protein, the first tau repeat domainlinked to the first reporter, and the second tau repeat domain linked tothe second reporter are stably expressed in the populations of cells. Insome such methods, nucleic acids encoding the Cas protein, the first taurepeat domain linked to the first reporter, and the second tau repeatdomain linked to the second reporter are genomically integrated in thepopulations of cells.

In some such methods, the cells are eukaryotic cells. Optionally, thecells are mammalian cells. Optionally, the cells are human cells.Optionally, the cells are HEK293T cells.

In some such methods, the plurality of unique guide RNAs are introducedat a concentration selected such that a majority of the cells receiveonly one of the unique guide RNAs. In some such methods, the pluralityof unique guide RNAs target 100 or more genes, 1000 or more genes, or10000 or more genes. In some such methods, the library is a genome-widelibrary. In some such methods, a plurality of target sequences aretargeted on average in each of the targeted plurality of genes.Optionally, at least three target sequences are targeted on average ineach of the targeted plurality of genes. Optionally, about three toabout six target sequences (e.g., about three, about four, or about six)are targeted on average in each of the targeted plurality of genes.

In some such methods, each guide RNA targets a constitutive exon.Optionally, each guide RNA targets a 5′ constitutive exon. In some suchmethods, each guide RNA targets a first exon, a second exon, or a thirdexon.

In some such methods, the plurality of unique guide RNAs are introducedinto the populations of cells by viral transduction. Optionally, each ofthe plurality of unique guide RNAs is in a separate viral vector. Insome such methods, the plurality of unique guide RNAs are introducedinto the populations of cells by lentiviral transduction. In some suchmethods, the populations of cells are infected at a multiplicity ofinfection of less than about 0.3.

In some such methods, the plurality of unique guide RNAs are introducedinto the populations of cells together with a selection marker, and step(b) further comprises selecting cells that comprise the selectionmarker. Optionally, the selection marker imparts resistance to a drug.Optionally, the selection marker imparts resistance to puromycin orzeocin. Optionally, the selection marker is selected from neomycinphosphotransferase, hygromycin B phosphotransferase,puromycin-N-acetyltransferase, and blasticidin S deaminase. Optionally,the selection marker is selected from neomycin phosphotransferase,hygromycin B phosphotransferase, puromycin-N-acetyltransferase,blasticidin S deaminase, and bleomycin resistance protein.

In some such methods, the populations of cells into which the pluralityof unique guide RNAs are introduced in step (b) each comprise greaterthan about 500 cells per unique guide RNA.

In some such methods, the time course in step (c) is more than about 1week. Optionally, the time course in step (c) is more than about 2weeks. In some such methods, the time course in step (c) comprises about10 to about 15 cell doublings. In some such methods, the plurality oftime points in step (c) comprises at least three time points.Optionally, the plurality of time points in step (c) comprises aboutfour time points or about six time points. In some such methods, thereis more than about 1 day between each time point in step (c).Optionally, there is more than about 2 days between each time point instep (c). Optionally, there is between about 3 to about 4 days betweeneach time point in step (c).

In some such methods, a gene is considered to exhibit syntheticlethality with tau protein aggregates in step (c) (or is expected toexhibit synthetic lethality with tau protein aggregates) if a guide RNAtargeting the gene is depleted in the aggregation-positive population ofcells but not in the aggregation-negative population of cells.Optionally, a gene is considered to exhibit synthetic lethality with tauprotein aggregates in step (c) (or is expected to exhibit syntheticlethality with tau protein aggregates) if a guide RNA targeting the genehas a more dramatic depletion pattern over the time course in theaggregation-positive population of cells relative to theaggregation-negative population of cells.

In some such methods, a guide RNA is considered depleted in step (c) ifthe abundance of the guide RNA at each time point is less than or equalto the abundance of the guide RNA at the preceding time point. In somesuch methods, a guide RNA is considered depleted in step (c) if theabundance of the guide RNA at each time point after the second timepoint is less than or equal to the abundance of the time point two timepoints prior.

In some such methods, a gene is considered to exhibit syntheticlethality with tau protein aggregates in step (c) (or is expected toexhibit synthetic lethality with tau protein aggregates) if more thanabout 30% of the guide RNAs in the library that target the gene aredepleted in the aggregation-positive population of cells but not in theaggregation-negative population of cells. Optionally, a gene isconsidered to exhibit synthetic lethality with tau protein aggregates instep (c) (or is expected to exhibit synthetic lethality with tau proteinaggregates) in any one of the following situations: (1) there is oneguide RNA in the library that targets the gene, and the one guide RNA isdepleted in the aggregation-positive population of cells but not in theaggregation-negative population of cells; (2) there are two guide RNAsin the library that target the gene, and at least one of the two guideRNAs is depleted in the aggregation-positive population of cells but notin the aggregation-negative population of cells; (3) there are threeguide RNAs in the library that target the gene, and at least one of thethree guide RNAs is depleted in the aggregation-positive population ofcells but not in the aggregation-negative population of cells; (4) thereare four guide RNAs in the library that target the gene, and at leasttwo of the four guide RNAs is depleted in the aggregation-positivepopulation of cells but not in the aggregation-negative population ofcells; (5) there are five guide RNAs in the library that target thegene, and at least two of the five guide RNAs is depleted in theaggregation-positive population of cells but not in theaggregation-negative population of cells; and (6) there are six guideRNAs in the library that target the gene, and at least three of the sixguide RNAs is depleted in the aggregation-positive population of cellsbut not in the aggregation-negative population of cells.

In some such methods, the method is repeated at least three times in atleast three different experiments, and a gene is considered to exhibitsynthetic lethality with tau protein aggregates (or is expected toexhibit synthetic lethality with tau protein aggregates) if it isconsidered to exhibit synthetic lethality with tau protein aggregates inmore than about 50% of the at least three different experiments.

In some such methods, the time course in step (c) is more than about 2weeks, wherein the plurality of time points in step (c) comprises aboutsix time points, wherein there is between about 3 to about 4 daysbetween each time point in step (c), wherein a guide RNA is considereddepleted in step (c) if the abundance of the guide RNA at each timepoint after the second time point is less than or equal to the abundanceof the time point two time points prior, and wherein a gene isconsidered to exhibit synthetic lethality with tau protein aggregates instep (c) (or is expected to exhibit synthetic lethality with tau proteinaggregates) if more than about 30% of the guide RNAs in the library thattarget the gene are depleted in the aggregation-positive population ofcells but not in the aggregation-negative population of cells.

Other such methods utilize CRISPR activation (CRISPR/Cas synergisticactivation mediator (SAM)) guide RNA libraries. Some such methodscomprise: (a) providing an aggregation-positive population of cells andan aggregation-negative population of cells, wherein each population ofcells comprises a chimeric Cas protein comprising a nuclease-inactiveCas protein (i.e., catalytically inactive Cas protein) fused to one ormore transcriptional activation domains, a chimeric adaptor proteincomprising an adaptor protein fused to one or more transcriptionalactivation domains, a first tau repeat domain linked to a firstreporter, and a second tau repeat domain linked to a second reporter,wherein in the aggregation-positive population of cells the first taurepeat domain linked to the first reporter and the second tau repeatdomain linked to the second reporter stably present in an aggregatedstate, and wherein in the aggregation-negative population of cells thefirst tau repeat domain linked to the first reporter and the second taurepeat domain linked to the second reporter do not stably present in anaggregated state; (b) introducing into each population of cells alibrary comprising a plurality of unique guide RNAs that target aplurality of genes, wherein the plurality of unique guide RNAs formcomplexes with the chimeric Cas protein and the chimeric adaptorprotein, and the complexes activate transcription of the plurality ofgenes resulting in increased gene expression; and (c) determiningabundance of each of the plurality of unique guide RNAs at a pluralityof time points over a time course in each population of cells, whereindepletion of a guide RNA in the aggregation-positive population of cellsbut not in the aggregation-negative population of cells or a moredramatic depletion pattern of a guide RNA over the time course in theaggregation-positive population of cells relative to theaggregation-negative population of cells indicates that activation ofthe gene targeted by the guide RNA exhibits synthetic lethality with tauprotein aggregates and is a genetic vulnerability associated with tauaggregation or is a candidate genetic vulnerability associated tauaggregation (e.g., for further testing via secondary screens).

In some such methods, the first tau repeat domain and/or the second taurepeat domain is a human tau repeat domain. In some such methods, thefirst tau repeat domain and/or the second tau repeat domain comprises apro-aggregation mutation. Optionally, the first tau repeat domain and/orthe second tau repeat domain comprises a tau P301S mutation. In somesuch methods, the first tau repeat domain and/or the second tau repeatdomain comprises a tau four-repeat domain. In some such methods, thefirst tau repeat domain and/or the second tau repeat domain comprisesSEQ ID NO: 11. In some such methods, the first tau repeat domain and thesecond tau repeat domain are the same. In some such methods, the firsttau repeat domain and the second tau repeat domain are the same and eachcomprises tau four-repeat domain comprising a tau P301S mutation.

In some such methods, the first reporter and the second reporter arefluorescent proteins. Optionally, the first reporter and the secondreporter are a fluorescence resonance energy transfer (FRET) pair.Optionally, the first reporter is cyan fluorescent protein (CFP) and thesecond reporter is yellow fluorescent protein (YFP).

In some such methods, the Cas protein is a Cas9 protein. Optionally, theCas protein is Streptococcus pyogenes Cas9. In some such methods, thechimeric Cas protein comprises the nuclease-inactive Cas protein fusedto a VP64 transcriptional activation domain, optionally wherein thechimeric Cas protein comprises from N-terminus to C-terminus: thenuclease-inactive Cas protein; a nuclear localization signal; and theVP64 transcriptional activator domain. In some such methods, the adaptorprotein is an MS2 coat protein, and the one or more transcriptionalactivation domains in the chimeric adaptor protein comprise a p65transcriptional activation domain and an HSF1 transcriptional activationdomain, optionally wherein the chimeric adaptor protein comprises fromN-terminus to C-terminus: the MS2 coat protein; a nuclear localizationsignal; the p65 transcriptional activation domain; and the HSF1transcriptional activation domain. In some such methods, the chimericCas protein comprises SEQ ID NO: 36, optionally wherein the chimeric Casprotein is encoded by a coding sequence comprising the sequence setforth in SEQ ID NO: 38. In some such methods, the chimeric adaptorprotein comprises SEQ ID NO: 37, optionally wherein the chimeric adaptorprotein is encoded by a coding sequence comprising the sequence setforth in SEQ ID NO: 39.

In some such methods, the chimeric Cas protein, the chimeric adaptorprotein, the first tau repeat domain linked to the first reporter, andthe second tau repeat domain linked to the second reporter are stablyexpressed in the population of cells. In some such methods, nucleicacids encoding the chimeric Cas protein, the chimeric adaptor protein,the first tau repeat domain linked to the first reporter, and the secondtau repeat domain linked to the second reporter are genomicallyintegrated in the populations of cells.

In some such methods, the cells are eukaryotic cells. Optionally, thecells are mammalian cells. Optionally, the cells are human cells.Optionally, the cells are HEK293T cells.

In some such methods, the plurality of unique guide RNAs are introducedat a concentration selected such that a majority of the cells receiveonly one of the unique guide RNAs. In some such methods, the pluralityof unique guide RNAs target 100 or more genes, 1000 or more genes, or10000 or more genes. In some such methods, the library is a genome-widelibrary. In some such methods, a plurality of target sequences aretargeted on average in each of the targeted plurality of genes.Optionally, at least three target sequences are targeted on average ineach of the targeted plurality of genes. Optionally, about three toabout six target sequences (e.g., about three, about four, or about six)are targeted on average in each of the targeted plurality of genes.Optionally, about three target sequences are targeted on average in eachof the targeted plurality of genes

In some such methods, each guide RNA targets a guide RNA target sequencewithin 200 bp upstream of a transcription start site. In some suchmethods, each guide RNA comprises one or more adaptor-binding elementsto which the chimeric adaptor protein can specifically bind. Optionally,each guide RNA comprises two adaptor-binding elements to which thechimeric adaptor protein can specifically bind. Optionally, a firstadaptor-binding element is within a first loop of each of the one ormore guide RNAs, and a second adaptor-binding element is within a secondloop of each of the one or more guide RNAs. Optionally, theadaptor-binding element comprises the sequence set forth in SEQ ID NO:33. Optionally, each of one or more guide RNAs is a single guide RNAcomprising a CRISPR RNA (crRNA) portion fused to a transactivatingCRISPR RNA (tracrRNA) portion, and the first loop is the tetraloopcorresponding to residues 13-16 of SEQ ID NO: 17, 19, 30, or 31, and thesecond loop is the stem loop 2 corresponding to residues 53-56 of SEQ IDNO: 17, 19, 30, or 31.

In some such methods, the plurality of unique guide RNAs are introducedinto the populations of cells by viral transduction. Optionally, each ofthe plurality of unique guide RNAs is in a separate viral vector. Insome such methods, the plurality of unique guide RNAs are introducedinto the populations of cells by lentiviral transduction. In some suchmethods, the populations of cells are infected at a multiplicity ofinfection of less than about 0.3.

In some such methods, the plurality of unique guide RNAs are introducedinto the populations of cells together with a selection marker, and step(b) further comprises selecting cells that comprise the selectionmarker. Optionally, the selection marker imparts resistance to a drug.Optionally, the selection marker imparts resistance to puromycin orzeocin. Optionally, the selection marker is selected from neomycinphosphotransferase, hygromycin B phosphotransferase,puromycin-N-acetyltransferase, and blasticidin S deaminase. Optionally,the selection marker is selected from neomycin phosphotransferase,hygromycin B phosphotransferase, puromycin-N-acetyltransferase,blasticidin S deaminase, and bleomycin resistance protein.

In some such methods, the populations of cells into which the pluralityof unique guide RNAs are introduced in step (b) each comprise greaterthan about 500 cells per unique guide RNA.

In some such methods, the time course in step (c) is more than about 1week. Optionally, the time course in step (c) is more than about 2weeks. In some such methods, the time course in step (c) comprises about10 to about 15 cell doublings. In some such methods, the plurality oftime points in step (c) comprises at least three time points.Optionally, the plurality of time points in step (c) comprises aboutfour time points or about six time points. In some such methods, thereis more than about 1 day between each time point in step (c).Optionally, there is more than about 2 days between each time point instep (c). Optionally, there is between about 3 to about 4 days betweeneach time point in step (c).

In some such methods, a gene is considered to exhibit syntheticlethality with tau protein aggregates in step (c) (or is expected toexhibit synthetic lethality with tau protein aggregates) if a guide RNAtargeting the gene is depleted in the aggregation-positive population ofcells but not in the aggregation-negative population of cells.Optionally, a gene is considered to exhibit synthetic lethality with tauprotein aggregates in step (c) (or is expected to exhibit syntheticlethality with tau protein aggregates) if a guide RNA targeting the genehas a more dramatic depletion pattern over the time course in theaggregation-positive population of cells relative to theaggregation-negative population of cells.

In some such methods, a guide RNA is considered depleted in step (c) ifthe abundance of the guide RNA at each time point is less than or equalto the abundance of the guide RNA at the preceding time point. In somesuch methods, a guide RNA is considered depleted in step (c) if theabundance of the guide RNA at each time point after the second timepoint is less than or equal to the abundance of the time point two timepoints prior.

In some such methods, a gene is considered to exhibit syntheticlethality with tau protein aggregates in step (c) (or is expected toexhibit synthetic lethality with tau protein aggregates) if more thanabout 30% of the guide RNAs in the library that target the gene aredepleted in the aggregation-positive population of cells but not in theaggregation-negative population of cells. Optionally, a gene isconsidered to exhibit synthetic lethality with tau protein aggregates instep (c) (or is expected to exhibit synthetic lethality with tau proteinaggregates) in any one of the following situations: (1) there is oneguide RNA in the library that targets the gene, and the one guide RNA isdepleted in the aggregation-positive population of cells but not in theaggregation-negative population of cells; (2) there are two guide RNAsin the library that target the gene, and at least one of the two guideRNAs is depleted in the aggregation-positive population of cells but notin the aggregation-negative population of cells; (3) there are threeguide RNAs in the library that target the gene, and at least one of thethree guide RNAs is depleted in the aggregation-positive population ofcells but not in the aggregation-negative population of cells; (4) thereare four guide RNAs in the library that target the gene, and at leasttwo of the four guide RNAs is depleted in the aggregation-positivepopulation of cells but not in the aggregation-negative population ofcells; (5) there are five guide RNAs in the library that target thegene, and at least two of the five guide RNAs is depleted in theaggregation-positive population of cells but not in theaggregation-negative population of cells; and (6) there are six guideRNAs in the library that target the gene, and at least three of the sixguide RNAs is depleted in the aggregation-positive population of cellsbut not in the aggregation-negative population of cells.

In some such methods, the method is repeated at least three times in atleast three different experiments, and a gene is considered to exhibitsynthetic lethality with tau protein aggregates (or is expected toexhibit synthetic lethality with tau protein aggregates) if it isconsidered to exhibit synthetic lethality with tau protein aggregates inmore than about 50% of the at least three different experiments.

In some such methods, the time course in step (c) is more than about 2weeks, wherein the plurality of time points in step (c) comprises aboutsix time points, wherein there is between about 3 to about 4 daysbetween each time point in step (c), wherein a guide RNA is considereddepleted in step (c) if the abundance of the guide RNA at each timepoint after the second time point is less than or equal to the abundanceof the time point two time points prior, and wherein a gene isconsidered to exhibit synthetic lethality with tau protein aggregates instep (c) (or is expected to exhibit synthetic lethality with tau proteinaggregates) if more than about 30% of the guide RNAs in the library thattarget the gene are depleted in the aggregation-positive population ofcells but not in the aggregation-negative population of cells.

In another aspect, provided are Cas-tau biosensor cells and populationof such cells such as a population of one or more cells comprising a Casprotein, a first tau repeat domain linked to a first reporter, and asecond tau repeat domain linked to a second reporter.

In some such cells, the first tau repeat domain and/or the second taurepeat domain is a human tau repeat domain. In some such cells, thefirst tau repeat domain and/or the second tau repeat domain comprises apro-aggregation mutation. Optionally, the first tau repeat domain and/orthe second tau repeat domain comprises a tau P301S mutation. In somesuch cells, the first tau repeat domain and/or the second tau repeatdomain comprises a tau four-repeat domain. In some such cells, the firsttau repeat domain and/or the second tau repeat domain comprises SEQ IDNO: 11. In some such cells, the first tau repeat domain and the secondtau repeat domain are the same. In some such cells, the first tau repeatdomain and the second tau repeat domain are the same and each comprisestau four-repeat domain comprising a tau P301S mutation.

In some such cells, the first reporter and the second reporter arefluorescent proteins. Optionally, the first reporter and the secondreporter are a fluorescence resonance energy transfer (FRET) pair.Optionally, the first reporter is cyan fluorescent protein (CFP) and thesecond reporter is yellow fluorescent protein (YFP).

In some such cells, the Cas protein is a Cas9 protein. Optionally, theCas protein is Streptococcus pyogenes Cas9. Optionally, the Cas proteincomprises SEQ ID NO: 21. Optionally, the Cas protein is encoded by acoding sequence comprising the sequence set forth in SEQ ID NO: 22.

In some such cells, the Cas protein, the first tau repeat domain linkedto the first reporter, and the second tau repeat domain linked to thesecond reporter are stably expressed in the cell. In some such cells,nucleic acids encoding the Cas protein, the first tau repeat domainlinked to the first reporter, and the second tau repeat domain linked tothe second reporter are genomically integrated in the cell.

Some such cells are eukaryotic cells. Optionally, the cells aremammalian cells. Optionally, the cells are human cells. Optionally, thecells are HEK293T cells. Optionally, the cells are in vitro.

In some such cells, the first tau repeat domain linked to the firstreporter and the second tau repeat domain linked to the second reporterare not stably present in an aggregated state. In some such cells, thefirst tau repeat domain linked to the first reporter and the second taurepeat domain linked to the second reporter stably present in anaggregated state.

In another aspect, provided are SAM-tau biosensor cells and populationof such cells such as a population of one or more cells comprising achimeric Cas protein comprising a nuclease-inactive Cas protein (i.e.,catalytically inactive Cas protein) fused to one or more transcriptionalactivation domains, a chimeric adaptor protein comprising an adaptorprotein fused to one or more transcriptional activation domains, a firsttau repeat domain linked to a first reporter, and a second tau repeatdomain linked to a second reporter.

In some such cells, the first tau repeat domain and/or the second taurepeat domain is a human tau repeat domain. In some such cells, thefirst tau repeat domain and/or the second tau repeat domain comprises apro-aggregation mutation. Optionally, the first tau repeat domain and/orthe second tau repeat domain comprises a tau P301S mutation. In somesuch cells, the first tau repeat domain and/or the second tau repeatdomain comprises a tau four-repeat domain. In some such cells, the firsttau repeat domain and/or the second tau repeat domain comprises SEQ IDNO: 11. In some such cells, the first tau repeat domain and the secondtau repeat domain are the same. In some such cells, the first tau repeatdomain and the second tau repeat domain are the same and each comprisestau four-repeat domain comprising a tau P301S mutation.

In some such cells, the first reporter and the second reporter arefluorescent proteins. Optionally, the first reporter and the secondreporter are a fluorescence resonance energy transfer (FRET) pair.Optionally, the first reporter is cyan fluorescent protein (CFP) and thesecond reporter is yellow fluorescent protein (YFP).

In some such cells, the Cas protein is a Cas9 protein. Optionally, theCas protein is Streptococcus pyogenes Cas9. In some such cells, thechimeric Cas protein comprises the nuclease-inactive Cas protein fusedto a VP64 transcriptional activation domain, optionally wherein thechimeric Cas protein comprises from N-terminus to C-terminus: thenuclease-inactive Cas protein; a nuclear localization signal; and theVP64 transcriptional activator domain. In some such cells, the adaptorprotein is an MS2 coat protein, and the one or more transcriptionalactivation domains in the chimeric adaptor protein comprise a p65transcriptional activation domain and an HSF1 transcriptional activationdomain, optionally wherein the chimeric adaptor protein comprises fromN-terminus to C-terminus: the MS2 coat protein; a nuclear localizationsignal; the p65 transcriptional activation domain; and the HSF1transcriptional activation domain. In some such cells, the chimeric Casprotein comprises SEQ ID NO: 36, optionally wherein the chimeric Casprotein is encoded by a coding sequence comprising the sequence setforth in SEQ ID NO: 38. In some such cells, the chimeric adaptor proteincomprises SEQ ID NO: 37, optionally wherein the chimeric adaptor proteinis encoded by a coding sequence comprising the sequence set forth in SEQID NO: 39.

In some such cells, the chimeric Cas protein, the chimeric adaptorprotein, the first tau repeat domain linked to the first reporter, andthe second tau repeat domain linked to the second reporter are stablyexpressed in the cell. In some such cells, nucleic acids encoding thechimeric Cas protein, the chimeric adaptor protein, the first tau repeatdomain linked to the first reporter, and the second tau repeat domainlinked to the second reporter are genomically integrated in the cell.

Some such cells are eukaryotic cells. Optionally, the cells aremammalian cells. Optionally, the cells are human cells. Optionally, thecells are HEK293T cells. Optionally, the cells are in vitro.

In some such cells, the first tau repeat domain linked to the firstreporter and the second tau repeat domain linked to the second reporterare not stably present in an aggregated state. In some such cells, thefirst tau repeat domain linked to the first reporter and the second taurepeat domain linked to the second reporter stably present in anaggregated state.

In another aspect, provided are in vitro cultures comprising any of theabove populations of cells or any of the cells disclosed herein and aculture medium.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 (not to scale) shows a schematic of tau isoform 2N4R. The taubiosensor lines include only tau4RD-YFP and tau4RD-CFP as transgenes,not the full 2N4R.

FIG. 2 shows a schematic of how aggregate formation is monitored byfluorescence resonance energy transfer (FRET) in Tau biosensor celllines. The Tau^(4RD)-CFP protein is excited by violet light and emitsblue light. The Tau^(4RD)-YFP fusion protein is excited by blue lightand emits yellow light. If there is no aggregation, excitation by violetlight will not lead to FRET. If there is Tau aggregation, excitation byviolet light will lead to FRET and yellow light emission.

FIG. 3A shows relative Cas9 mRNA expression inTau^(4RD)-CFP/Tau^(4RD)-YFP (TCY) biosensor cell clones transduced withlentiviral Cas9 expression constructs relative to clone Cas9H1, which isa control underperforming Cas9-expression TCY clone that was previouslyisolated.

FIG. 3B shows cutting efficiency at the PERK locus and the SNCA locus inthe Cas9 TCY clones three and seven days after transduction with sgRNAstargeting PERK and SNCA respectively.

FIG. 4 shows a schematic of the strategy for disruption of target genesin Cas9 TCY biosensor cell using a genome-wide CRISPR/Cas9 sgRNAlibrary.

FIG. 5 is a schematic showing derivation ofTau^(4RD)-CFP/Tau^(4RD)-YFP/Cas9 Agg[+] subclones containing stablypropagating Tau aggregates. A FRET microscopy image showing the subclonewith Tau aggregates is also shown.

FIG. 6 is a schematic of an experiment to assess genes perturbed by Tauaggregation.

FIG. 7 shows results from unsupervised clustering that indicate thatAgg[+] clone samples and Agg[−] clone samples are distinct byaggregation status (clustering procedure: log transformation of readsper kilobase million (RPKM); distance metric: 1−absolute value ofPearson correlation coefficient; hierarchical clustering).

FIG. 8 shows genes significantly perturbed by aggregation in Tau Agg[+]cells (significantly perturbed genes defined as fold change ≥1.5 (eitherup or down) and p value ≤0.01.

FIG. 9 shows a general schematic of the pooled genome-wide CRISPRnuclease (CRISPRn) screen to reveal genetic vulnerabilities associatedwith Tau aggregation.

FIG. 10A shows a general schematic of the identification of essentialgenes (sgRNAs targeting those genes that are depleted in both Agg[+] andAgg[−]).

FIG. 10B shows a general schematic of the identification of syntheticlethal genes (sgRNAs targeting those genes that are depleted in Agg[+]but not in Agg[−]).

FIG. 11 shows a schematic of the full time course approach to identifygenes for which there is a sgRNA depletion pattern over time.

FIG. 12 shows the number of genes for which sgRNAs were depleted inAgg[+] and Agg[−] samples.

FIG. 13 is a graph showing the overlap of essential genes identified inthe Tau biosensor screen compared to other published reports.

FIG. 14 is a graph showing the identification of 71 genes with uniquelydepleted sgRNAs over time in comparison to Day 3 in Agg[+] samplescompared to Agg[−] samples in three repeat experiments.

FIGS. 15A and 15B show the fold change over time (sgRNA depletion)relative to Day 3 in Agg[+] and Agg[−] cells for sgRNAs targeting threecandidate essential genes (Essential Genes 6, 8, and 9 (EG6, EG8, andEG9); FIG. 15A) and three candidate synthetic lethal genes (Target Genes1, 5, and 6 (TG1, TG5, and TG6); FIG. 15B).

FIG. 16 shows read count distribution of the custom sgRNA libraryconsisting of 462 unique targeting sgRNAs targeting 71 putativesynthetic lethal genes (for which sgRNAs were selectively depleted inAgg[+] cells in the primary screening) and 10 putative essential genes(for which sgRNAs were depleted in both Agg[+] and Agg[−] cells in theprimary screening).

FIG. 17 shows read count distribution of a sgRNA library consisting of500 unique non-targeting control sgRNAs (non-specific sgRNAs that werenot altered over time in the primary screening).

FIG. 18 shows a general schematic of the secondary screen to validatethe 71 putative synthetic lethal genes and the 10 putative essentialgenes using the custom sgRNA libraries.

FIGS. 19A-19C show the fold change over time (sgRNA depletion) relativeto Day 3 in Agg[+] and Agg[−] cells for sgRNAs targeting ten candidateessential genes (EG1-EG10) in the secondary screen.

FIG. 20 shows an example of a graph showing a distinguishable depletionpattern for a sgRNA between Agg[+] cells and Agg[−] cells (i.e., moredramatic depletion in Agg[+] cells).

FIG. 21 shows a general schematic for identification of synthetic lethalgenes in the secondary screen by identifying sgRNAs that demonstrateboth a significant time course p-value and a depleting pattern in everyexperiment (Day 10<Day 3, Day 14<Day 7, and Day 17<Day 10).

FIG. 22A shows the fold change over time (sgRNA depletion) in Agg[+] andAgg[−] cells for six sgRNAs targeting Target Gene 1 (TG1) and six sgRNAsTarget Gene 2 (TG2).

FIG. 22B shows the fold change over time (sgRNA depletion) in Agg[+] andAgg[−] cells for six sgRNAs targeting Target Gene 3 (TG3) and six sgRNAsTarget Gene 4 (TG4).

FIG. 23 shows a general schematic for identification of synthetic lethalgenes in the secondary screen by identifying CRISPRa sgRNAs thatdemonstrate both a significant time course p-value and a depletingpattern in every experiment (Day 10<Day 3, Day 14<Day 7, and Day 17<Day10).

FIGS. 24A and 24B show the fold change over time (sgRNA depletion) inAgg[+] and Agg[−] cells for sgRNAs targeting Target Genes 7-15(TG7-TG15).

FIG. 25 shows a schematic for further validating Target Genes 1, 2, and4, using flow cytometry to quantitate cell viability and cell death inAgg[+] cells and Agg[−] cells following transduction with lentiviralvectors delivering sgRNAs targeting Target Genes 1, 2, and 4.

FIG. 26 shows cell viability and cell death in Agg[+] cells and Agg[−]cells following transduction with lentiviral vectors delivering sgRNAstargeting Target Genes 1, 2, and 4.

DEFINITIONS

The terms “protein,” “polypeptide,” and “peptide,” used interchangeablyherein, include polymeric forms of amino acids of any length, includingcoded and non-coded amino acids and chemically or biochemically modifiedor derivatized amino acids. The terms also include polymers that havebeen modified, such as polypeptides having modified peptide backbones.The term “domain” refers to any part of a protein or polypeptide havinga particular function or structure.

Proteins are said to have an “N-terminus” and a “C-terminus.” The term“N-terminus” relates to the start of a protein or polypeptide,terminated by an amino acid with a free amine group (—NH₂). The term“C-terminus” relates to the end of an amino acid chain (protein orpolypeptide), terminated by a free carboxyl group (—COOH).

The terms “nucleic acid” and “polynucleotide,” used interchangeablyherein, include polymeric forms of nucleotides of any length, includingribonucleotides, deoxyribonucleotides, or analogs or modified versionsthereof. They include single-, double-, and multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purinebases, pyrimidine bases, or other natural, chemically modified,biochemically modified, non-natural, or derivatized nucleotide bases.

Nucleic acids are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. An end of an oligonucleotide is referred to as the “5′ end” ifits 5′ phosphate is not linked to the 3′ oxygen of a mononucleotidepentose ring. An end of an oligonucleotide is referred to as the “3′end” if its 3′ oxygen is not linked to a 5′ phosphate of anothermononucleotide pentose ring. A nucleic acid sequence, even if internalto a larger oligonucleotide, also may be said to have 5′ and 3′ ends. Ineither a linear or circular DNA molecule, discrete elements are referredto as being “upstream” or 5′ of the “downstream” or 3′ elements.

The term “genomically integrated” refers to a nucleic acid that has beenintroduced into a cell such that the nucleotide sequence integrates intothe genome of the cell. Any protocol may be used for the stableincorporation of a nucleic acid into the genome of a cell.

The term “targeting vector” refers to a recombinant nucleic acid thatcan be introduced by homologous recombination,non-homologous-end-joining-mediated ligation, or any other means ofrecombination to a target position in the genome of a cell.

The term “viral vector” refers to a recombinant nucleic acid thatincludes at least one element of viral origin and includes elementssufficient for or permissive of packaging into a viral vector particle.The vector and/or particle can be utilized for the purpose oftransferring DNA, RNA, or other nucleic acids into cells either ex vivoor in vivo. Numerous forms of viral vectors are known.

The term “wild type” includes entities having a structure and/oractivity as found in a normal (as contrasted with mutant, diseased,altered, or so forth) state or context. Wild type genes and polypeptidesoften exist in multiple different forms (e.g., alleles).

The term “endogenous sequence” refers to a nucleic acid sequence thatoccurs naturally within a cell or organism. For example, an endogenousMAPT sequence of a cell or organism refers to a native MAPT sequencethat naturally occurs at the MAPT locus in the cell or organism.

“Exogenous” molecules or sequences include molecules or sequences thatare not normally present in a cell in that form. Normal presenceincludes presence with respect to the particular developmental stage andenvironmental conditions of the cell. An exogenous molecule or sequence,for example, can include a mutated version of a corresponding endogenoussequence within the cell, such as a humanized version of the endogenoussequence, or can include a sequence corresponding to an endogenoussequence within the cell but in a different form (i.e., not within achromosome). In contrast, endogenous molecules or sequences includemolecules or sequences that are normally present in that form in aparticular cell at a particular developmental stage under particularenvironmental conditions.

The term “heterologous” when used in the context of a nucleic acid or aprotein indicates that the nucleic acid or protein comprises at leasttwo segments that do not naturally occur together in the same molecule.For example, the term “heterologous,” when used with reference tosegments of a nucleic acid or segments of a protein, indicates that thenucleic acid or protein comprises two or more sub-sequences that are notfound in the same relationship to each other (e.g., joined together) innature. As one example, a “heterologous” region of a nucleic acid vectoris a segment of nucleic acid within or attached to another nucleic acidmolecule that is not found in association with the other molecule innature. For example, a heterologous region of a nucleic acid vectorcould include a coding sequence flanked by sequences not found inassociation with the coding sequence in nature. Likewise, a“heterologous” region of a protein is a segment of amino acids within orattached to another peptide molecule that is not found in associationwith the other peptide molecule in nature (e.g., a fusion protein, or aprotein with a tag). Similarly, a nucleic acid or protein can comprise aheterologous label or a heterologous secretion or localization sequence.

The term “locus” refers to a specific location of a gene (or significantsequence), DNA sequence, polypeptide-encoding sequence, or position on achromosome of the genome of an organism. For example, a “MAPT locus” mayrefer to the specific location of a MAPT gene, MAPT DNA sequence,microtubule-associated-protein-tau-encoding sequence, or MAPT positionon a chromosome of the genome of an organism that has been identified asto where such a sequence resides. A “MAPT locus” may comprise aregulatory element of a MAPT gene, including, for example, an enhancer,a promoter, 5′ and/or 3′ untranslated region (UTR), or a combinationthereof.

The term “gene” refers to a DNA sequence in a chromosome that codes fora product (e.g., an RNA product and/or a polypeptide product) andincludes the coding region interrupted with non-coding introns andsequence located adjacent to the coding region on both the 5′ and 3′ends such that the gene corresponds to the full-length mRNA (includingthe 5′ and 3′ untranslated sequences). The term “gene” also includesother non-coding sequences including regulatory sequences (e.g.,promoters, enhancers, and transcription factor binding sites),polyadenylation signals, internal ribosome entry sites, silencers,insulating sequence, and matrix attachment regions. These sequences maybe close to the coding region of the gene (e.g., within 10 kb) or atdistant sites, and they influence the level or rate of transcription andtranslation of the gene.

The term “allele” refers to a variant form of a gene. Some genes have avariety of different forms, which are located at the same position, orgenetic locus, on a chromosome. A diploid organism has two alleles ateach genetic locus. Each pair of alleles represents the genotype of aspecific genetic locus. Genotypes are described as homozygous if thereare two identical alleles at a particular locus and as heterozygous ifthe two alleles differ.

A “promoter” is a regulatory region of DNA usually comprising a TATA boxcapable of directing RNA polymerase II to initiate RNA synthesis at theappropriate transcription initiation site for a particularpolynucleotide sequence. A promoter may additionally comprise otherregions which influence the transcription initiation rate. The promotersequences disclosed herein modulate transcription of an operably linkedpolynucleotide. A promoter can be active in one or more of the celltypes disclosed herein (e.g., a human cell, a pluripotent cell, aone-cell stage embryo, a differentiated cell, or a combination thereof).A promoter can be, for example, a constitutively active promoter, aconditional promoter, an inducible promoter, a temporally restrictedpromoter (e.g., a developmentally regulated promoter), or a spatiallyrestricted promoter (e.g., a cell-specific or tissue-specific promoter).Examples of promoters can be found, for example, in WO 2013/176772,herein incorporated by reference in its entirety for all purposes.

“Operable linkage” or being “operably linked” includes juxtaposition oftwo or more components (e.g., a promoter and another sequence element)such that both components function normally and allow the possibilitythat at least one of the components can mediate a function that isexerted upon at least one of the other components. For example, apromoter can be operably linked to a coding sequence if the promotercontrols the level of transcription of the coding sequence in responseto the presence or absence of one or more transcriptional regulatoryfactors. Operable linkage can include such sequences being contiguouswith each other or acting in trans (e.g., a regulatory sequence can actat a distance to control transcription of the coding sequence).

The term “variant” refers to a nucleotide sequence differing from thesequence most prevalent in a population (e.g., by one nucleotide) or aprotein sequence different from the sequence most prevalent in apopulation (e.g., by one amino acid).

The term “fragment” when referring to a protein means a protein that isshorter or has fewer amino acids than the full-length protein. The term“fragment” when referring to a nucleic acid means a nucleic acid that isshorter or has fewer nucleotides than the full-length nucleic acid. Afragment can be, for example, an N-terminal fragment (i.e., removal of aportion of the C-terminal end of the protein), a C-terminal fragment(i.e., removal of a portion of the N-terminal end of the protein), or aninternal fragment.

“Sequence identity” or “identity” in the context of two polynucleotidesor polypeptide sequences refers to the residues in the two sequencesthat are the same when aligned for maximum correspondence over aspecified comparison window. When percentage of sequence identity isused in reference to proteins, residue positions which are not identicaloften differ by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. When sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well known. Typically, this involves scoring aconservative substitution as a partial rather than a full mismatch,thereby increasing the percentage sequence identity. Thus, for example,where an identical amino acid is given a score of 1 and anon-conservative substitution is given a score of zero, a conservativesubstitution is given a score between zero and 1. The scoring ofconservative substitutions is calculated, e.g., as implemented in theprogram PC/GENE (Intelligenetics, Mountain View, California).

“Percentage of sequence identity” includes the value determined bycomparing two optimally aligned sequences (greatest number of perfectlymatched residues) over a comparison window, wherein the portion of thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) as compared to the reference sequence (whichdoes not comprise additions or deletions) for optimal alignment of thetwo sequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison, and multiplying the result by 100to yield the percentage of sequence identity. Unless otherwise specified(e.g., the shorter sequence includes a linked heterologous sequence),the comparison window is the full length of the shorter of the twosequences being compared.

Unless otherwise stated, sequence identity/similarity values include thevalue obtained using GAP Version 10 using the following parameters: %identity and % similarity for a nucleotide sequence using GAP Weight of50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; %identity and % similarity for an amino acid sequence using GAP Weight of8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or anyequivalent program thereof “Equivalent program” includes any sequencecomparison program that, for any two sequences in question, generates analignment having identical nucleotide or amino acid residue matches andan identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

The term “conservative amino acid substitution” refers to thesubstitution of an amino acid that is normally present in the sequencewith a different amino acid of similar size, charge, or polarity.Examples of conservative substitutions include the substitution of anon-polar (hydrophobic) residue such as isoleucine, valine, or leucinefor another non-polar residue. Likewise, examples of conservativesubstitutions include the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, or between glycine and serine. Additionally,the substitution of a basic residue such as lysine, arginine, orhistidine for another, or the substitution of one acidic residue such asaspartic acid or glutamic acid for another acidic residue are additionalexamples of conservative substitutions. Examples of non-conservativesubstitutions include the substitution of a non-polar (hydrophobic)amino acid residue such as isoleucine, valine, leucine, alanine, ormethionine for a polar (hydrophilic) residue such as cysteine,glutamine, glutamic acid or lysine and/or a polar residue for anon-polar residue. Typical amino acid categorizations are summarizedbelow.

TABLE 1 Amino Acid Categorizations. Alanine Ala A Nonpolar Neutral 1.8Arginine Arg R Polar Positive −4.5 Asparagine Asn N Polar Neutral −3.5Aspartic acid Asp D Polar Negative −3.5 Cysteine Cys C Nonpolar Neutral2.5 Glutamic acid Glu E Polar Negative −3.5 Glutamine Gln Q PolarNeutral −3.5 Glycine Gly G Nonpolar Neutral −0.4 Histidine His H PolarPositive −3.2 Isoleucine Ile I Nonpolar Neutral 4.5 Leucine Leu LNonpolar Neutral 3.8 Lysine Lys K Polar Positive −3.9 Methionine Met MNonpolar Neutral 1.9 Phenylalanine Phe F Nonpolar Neutral 2.8 ProlinePro P Nonpolar Neutral −1.6 Serine Ser S Polar Neutral −0.8 ThreonineThr T Polar Neutral −0.7 Tryptophan Trp W Nonpolar Neutral −0.9 TyrosineTyr Y Polar Neutral −1.3 Valine Val V Nonpolar Neutral 4.2

A “homologous” sequence (e.g., nucleic acid sequence) includes asequence that is either identical or substantially similar to a knownreference sequence, such that it is, for example, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical to the knownreference sequence. Homologous sequences can include, for example,orthologous sequence and paralogous sequences. Homologous genes, forexample, typically descend from a common ancestral DNA sequence, eitherthrough a speciation event (orthologous genes) or a genetic duplicationevent (paralogous genes). “Orthologous” genes include genes in differentspecies that evolved from a common ancestral gene by speciation.Orthologs typically retain the same function in the course of evolution.“Paralogous” genes include genes related by duplication within a genome.Paralogs can evolve new functions in the course of evolution.

The term “in vitro” includes artificial environments and to processes orreactions that occur within an artificial environment (e.g., a test tubeor an isolated cell or cell line). The term “in vivo” includes naturalenvironments (e.g., a cell or organism or body) and to processes orreactions that occur within a natural environment. The term “ex vivo”includes cells that have been removed from the body of an individual andto processes or reactions that occur within such cells.

The term “reporter gene” refers to a nucleic acid having a sequenceencoding a gene product (typically an enzyme) that is easily andquantifiably assayed when a construct comprising the reporter genesequence operably linked to a heterologous promoter and/or enhancerelement is introduced into cells containing (or which can be made tocontain) the factors necessary for the activation of the promoter and/orenhancer elements. Examples of reporter genes include, but are notlimited, to genes encoding beta-galactosidase (lacZ), the bacterialchloramphenicol acetyltransferase (cat) genes, firefly luciferase genes,genes encoding beta-glucuronidase (GUS), and genes encoding fluorescentproteins. A “reporter protein” refers to a protein encoded by a reportergene.

The term “fluorescent reporter protein” as used herein means a reporterprotein that is detectable based on fluorescence wherein thefluorescence may be either from the reporter protein directly, activityof the reporter protein on a fluorogenic substrate, or a protein withaffinity for binding to a fluorescent tagged compound. Examples offluorescent proteins include green fluorescent proteins (e.g., GFP,GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, Monomeric AzamiGreen, CopGFP, AceGFP, and ZsGreenl), yellow fluorescent proteins (e.g.,YFP, eYFP, Citrine, Venus, YPet, PhiYFP, and ZsYellowl), bluefluorescent proteins (e.g., BFP, eBFP, eBFP2, Azurite, mKalamal, GFPuv,Sapphire, and T-sapphire), cyan fluorescent proteins (e.g., CFP, eCFP,Cerulean, CyPet, AmCyanl, and Midoriishi-Cyan), red fluorescent proteins(e.g., RFP, mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP I,DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2,eqFP611, mRaspberry, mStrawberry, and Jred), orange fluorescent proteins(e.g., mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange,mTangerine, and tdTomato), and any other suitable fluorescent proteinwhose presence in cells can be detected by flow cytometry methods.

Repair in response to double-strand breaks (DSBs) occurs principallythrough two conserved DNA repair pathways: homologous recombination (HR)and non-homologous end joining (NHEJ). See Kasparek & Humphrey (2011)Seminars in Cell & Dev. Biol. 22:886-897, herein incorporated byreference in its entirety for all purposes. Likewise, repair of a targetnucleic acid mediated by an exogenous donor nucleic acid can include anyprocess of exchange of genetic information between the twopolynucleotides.

The term “recombination” includes any process of exchange of geneticinformation between two polynucleotides and can occur by any mechanism.Recombination can occur via homology directed repair (HDR) or homologousrecombination (HR). HDR or HR includes a form of nucleic acid repairthat can require nucleotide sequence homology, uses a “donor” moleculeas a template for repair of a “target” molecule (i.e., the one thatexperienced the double-strand break), and leads to transfer of geneticinformation from the donor to target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or synthesis-dependent strand annealing, in which the donor is usedto resynthesize genetic information that will become part of the target,and/or related processes. In some cases, the donor polynucleotide, aportion of the donor polynucleotide, a copy of the donor polynucleotide,or a portion of a copy of the donor polynucleotide integrates into thetarget DNA. See Wang et al. (2013) Cell 153:910-918; Mandalos et al.(2012) PLOS ONE 7:e45768:1-9; and Wang et al. (2013) Nat Biotechnol.31:530-532, each of which is herein incorporated by reference in itsentirety for all purposes.

NHEJ includes the repair of double-strand breaks in a nucleic acid bydirect ligation of the break ends to one another or to an exogenoussequence without the need for a homologous template. Ligation ofnon-contiguous sequences by NHEJ can often result in deletions,insertions, or translocations near the site of the double-strand break.For example, NHEJ can also result in the targeted integration of anexogenous donor nucleic acid through direct ligation of the break endswith the ends of the exogenous donor nucleic acid (i.e., NHEJ-basedcapture). Such NHEJ-mediated targeted integration can be preferred forinsertion of an exogenous donor nucleic acid when homology directedrepair (HDR) pathways are not readily usable (e.g., in non-dividingcells, primary cells, and cells which perform homology-based DNA repairpoorly). In addition, in contrast to homology-directed repair, knowledgeconcerning large regions of sequence identity flanking the cleavage siteis not needed, which can be beneficial when attempting targetedinsertion into organisms that have genomes for which there is limitedknowledge of the genomic sequence. The integration can proceed vialigation of blunt ends between the exogenous donor nucleic acid and thecleaved genomic sequence, or via ligation of sticky ends (i.e., having5′ or 3′ overhangs) using an exogenous donor nucleic acid that isflanked by overhangs that are compatible with those generated by anuclease agent in the cleaved genomic sequence. See, e.g., US2011/020722, WO 2014/033644, WO 2014/089290, and Maresca et al. (2013)Genome Res. 23(3):539-546, each of which is herein incorporated byreference in its entirety for all purposes. If blunt ends are ligated,target and/or donor resection may be needed to generation regions ofmicrohomology needed for fragment joining, which may create unwantedalterations in the target sequence.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” a protein maycontain the protein alone or in combination with other ingredients. Thetransitional phrase “consisting essentially of” means that the scope ofa claim is to be interpreted to encompass the specified elements recitedin the claim and those that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. Thus, the term “consistingessentially of” when used in a claim of this invention is not intendedto be interpreted to be equivalent to “comprising.”

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur and that the description includesinstances in which the event or circumstance occurs and instances inwhich it does not.

Designation of a range of values includes all integers within ordefining the range, and all subranges defined by integers within therange.

Unless otherwise apparent from the context, the term “about” encompassesvalues within a standard margin of error of measurement (e.g., SEM) of astated value.

The term “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

The term “or” refers to any one member of a particular list and alsoincludes any combination of members of that list.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a protein” or “at least one protein” can include a pluralityof proteins, including mixtures thereof.

Statistically significant means p ≤0.05.

DETAILED DESCRIPTION I. Overview

Cas-protein-ready tau biosensor cells and methods of making and usingsuch cells to screen for genetic vulnerability associated with tauaggregation are provided. CRISPR/Cas synergistic activation mediator(SAM)-ready tau biosensor cells and methods of making and using suchcells to screen for genetic vulnerability associated with tauaggregation are also provided.

To identify genes and pathways that exhibit synthetic lethality withdisease-associated protein aggregates, a platform was developed forperforming genome-wide screens with CRISPR nuclease (CRISPRn) sgRNAlibraries. The platform identifies genes that, when disrupted, causecell death specifically in the context of abnormal protein aggregation.To identify further genes and pathways that exhibit synthetic lethalitywith disease-associated protein aggregates, a platform was developed forperforming genome-wide screens with CRISPR activation (CRISPRa) sgRNAlibraries. The platform identifies genes that, when activated, causecell death specifically in the context of abnormal protein aggregation.The identification of such genes may elucidate the mechanisms ofaggregate-associated neurotoxicity, and genetic pathways that promotedeath of neurons in the context of neurodegenerative disease.

The screens employ a tau biosensor cell line (e.g., human cell line, orHEK293T) consisting of cells stably expressing tau repeat domain (e.g.,tau four-repeat domain, tau_4RD) with a pathogenic mutation (e.g., theP301S pathogenic mutation), linked to unique reporters that can acttogether as an intracellular biosensor that produces a detectable signalwhen aggregated. In one non-limiting example, the cell lines contain twotransgenes stably expressing disease-associated protein variants fusedto the fluorescent protein CFP (e.g., eCFP) or the fluorescent proteinYFP (e.g., eYFP): tau^(4RD)-CFP/tau^(4RD)-YFP (TCY), wherein the taurepeat domain (4RD) comprises the P301S pathogenic mutation. In thesebiosensor lines, tau-CFP/tau-YFP protein aggregation produces afluorescence resonance energy transfer (FRET) signal, the result of atransfer of fluorescent energy from donor CFP to acceptor YFP. The termCFP (cyan fluorescent protein) when used herein includes eCFP (enhancedcyan fluorescent protein), and the term YFP (yellow fluorescent protein)when used herein includes eYFP (enhanced yellow fluorescent protein).FRET-positive cells, which contain tau aggregates, can be sorted andisolated by flow cytometry. At baseline, unstimulated cells express thereporters in a stable, soluble state with minimal FRET signal. Uponstimulation (e.g., liposome transfection of seed particles), thereporter proteins form aggregates, producing a FRET signal.Aggregate-containing cells can be isolated by FACS. Stably propagatingaggregate-containing cell lines, Agg[+], can be isolated by clonalserial dilution of Agg[−] cell lines.

Several modifications were made to this tau biosensor cell line to makeit useful for genetic screening using CRISPRn libraries. First, thesetau biosensor cells were modified by introducing a Cas-expressingtransgene (e.g., Cas9 or SpCas9) for use in the CRISPRn screens. Second,subclones of these Cas-expressing tau biosensor lines were derived inwhich tau protein is stably present in either a non-aggregated (thedefault state) (Agg[−]) or an aggregated state (Agg[+]).

These cell lines were used to develop a method of screening in whichCas-expressing tau biosensor cells, either with aggregates (Agg[+]) orwithout aggregates (Agg[−]), were transduced with a CRISPR guide RNAlibrary to introduce knock-out mutations at each target gene. Screeningwas then done to identify not only essential genes, consisting of thosegenes whose targeting sgRNAs become depleted over time in both theAgg[+] and Agg[−] cell lines, but also synthetic lethal genes,consisting of genes whose targeting sgRNAs become depleted over timepreferentially in the Agg[+] as compared to the Agg[−] cell lines.Depletion profiles were assessed using a newly defined time courseanalysis, in which guide RNAs with a consistent pattern of decreasingreads from the earliest time point to the final time point wereconsidered to be depleted. This is a novel analytical approach toevaluating guide RNA depletion, as compared to the more conventionalapproach of simply comparing reads of the endpoint cell collection tothe first passage.

Likewise, several modifications were made to this tau biosensor cellline to make it useful for genetic screening using CRISPRa libraries(e.g., for use with a CRISPR/Cas synergistic activation mediator (SAM)system). In an exemplary SAM system, several activation domains interactto cause a greater transcriptional activation than could be induced byany one factor alone. For example, an exemplary SAM system comprises achimeric Cas protein comprising a nuclease-inactive Cas protein fused toone or more transcriptional activation domains (e.g., VP64) and achimeric adaptor protein comprising an adaptor protein (e.g., MS2 coatprotein (MCP)) fused to one or more transcriptional activation domains(e.g., fused to p65 and HSF1). The MCP naturally binds to MS2 stemloops. In an exemplary SAM system, MCP interacts MS2 stem loopsengineered into the CRISPR-associated sgRNA and thereby shuttles thebound transcription factors to the appropriate genomic location.

First, these tau biosensor cells were modified by introducing one ormore transgenes expressing the chimeric Cas protein comprising thenuclease-inactive Cas protein fused to one or more transcriptionalactivation domains (e.g., VP64) and the chimeric adaptor proteincomprising the adaptor protein (e.g., MS2 coat protein (MCP)) fused toone or more transcriptional activation domains (e.g., fused to p65 andHSF1). Although SAM systems are described herein, other CRISPRa systemssuch as a nuclease-inactive Cas protein fused to one or moretranscriptional activation domains, wherein such systems do not alsoinclude a chimeric adaptor protein, can also be used. In such cases, thetau biosensor cells would be modified by introducing a transgeneexpressing the chimeric Cas protein.

Second, subclones of these SAM-expressing (i.e., chimeric-Cas-expressingand chimeric-adaptor-expressing) tau biosensor lines were derived inwhich tau protein is stably present in either a non-aggregated (thedefault state) (Agg[−]) or an aggregated state (Agg[+]).

These cell lines were used to develop a method of screening in whichSAM-expressing tau biosensor cells, either with aggregates (Agg[+]) orwithout aggregates (Agg[−]), were transduced with a CRISPRa guide RNAlibrary to transcriptionally activate each target gene. Screening wasthen done to identify gene that when activated have a synthetic lethaleffect. Specifically, sgRNAs that are depleted specifically in theAgg[+] cell line, while not or less-depleted in the Agg[−] cell line,may indicate a synthetic lethal effect, in which the activation of aspecific target gene combines with the presence of Tau aggregates in thecell to induce cell death. Depletion profiles were assessed using anewly defined time course analysis, in which guide RNAs with aconsistent pattern of decreasing reads from the earliest time point tothe final time point were considered to be depleted. This is a novelanalytical approach to evaluating guide RNA depletion, as compared tothe more conventional approach of simply comparing reads of the endpointcell collection to the first passage. These synthetic lethal genes areof interest as potential modifiers of tau-associated cell toxicity.

II. Cas/Tau Biosensor and SAM/Tau Biosensor Cell Lines and Methods ofGenerating

A. Cas/Tau Biosensor Cells and SAM/Tau Biosensor Cells

Disclosed herein are cells not only expressing a first tau repeat domain(e.g., comprising the tau microtubule binding domain (MBD)) linked to afirst reporter and a second tau repeat domain linked to a secondreporter, but also expressing a Cas protein, such as Cas9. Alsodisclosed herein are cells not only expressing a first tau repeat domain(e.g., comprising the tau microtubule binding domain (MBD)) linked to afirst reporter and a second tau repeat domain linked to a secondreporter, but also expressing a chimeric Cas protein comprising anuclease-inactive Cas protein fused to one or more transcriptionalactivation domains and a chimeric adaptor protein comprising an adaptorprotein fused to one or more transcriptional activation domains. Thefirst tau repeat domain linked to the first reporter can be stablyexpressed, and the second tau repeat domain linked to the secondreporter can be stably expressed. For example, DNA encoding the firsttau repeat domain linked to the first reporter can be genomicallyintegrated, and DNA encoding the second tau repeat domain linked to thesecond reporter can be genomically integrated. Similarly, the Casprotein can be stably expressed in the Cas/tau biosensor cells. Forexample, DNA encoding the Cas protein can be genomically integrated.Likewise, the chimeric Cas protein and/or the chimeric adaptor proteincan be stably expressed in the SAM/tau biosensor cells. For example, DNAencoding the chimeric Cas protein can be genomically integrated and/orDNA encoding the chimeric adaptor protein can be genomically integrated.

1. Tau and Tau Repeat Domains Linked to Reporters

Microtubule-associated protein tau is a protein that promotesmicrotubule assembly and stability and is predominantly expressed inneurons. Tau has a role in stabilizing neuronal microtubules and thus inpromoting axonal outgrowth. In Alzheimer disease (AD) and a family ofrelated neurodegenerative diseases called tauopathies, tau protein isabnormally hyperphosphorylated and aggregated into bundles of filaments(paired helical filaments), which manifest as neurofibrillary tangles.Tauopathies are a group of heterogeneous neurodegenerative conditionscharacterized by deposition of abnormal tau in the brain.

The tau repeat domain can be from a tau protein from any animal ormammal, such as human, mouse, or rat. In one specific example, the taurepeat domain is from a human tau protein. An exemplary human tauprotein is assigned UniProt accession number P10636. The tau proteinsare the products of alternate splicing from a single gene that in humansis designated MAPT (microtubule-associated protein tau). The tau repeatdomain carries the sequence motifs responsible for aggregation (i.e., itis the aggregation-prone domain from tau). Depending on splicing, therepeat domain of the tau protein has either three or four repeat regionsthat constitute the aggregation-prone core of the protein, which isoften termed the repeat domain (RD). Specifically, the repeat domain oftau represents the core of the microtubule binding region and harborsthe hexapeptide motifs in R2 and R3 that are responsible for Tauaggregation. In the human brain, there are six tau isoforms ranging from352 to 441 amino acids in length. These isoforms vary at the carboxylterminal according to the presence of either three repeat or four repeatdomains (R1-R4), in addition to the presence or absence of one or twoinsert domains at the amino-terminus. The repeat domains, located at thecarboxyl-terminal half of tau, are believed to be important formicrotubule binding as well as for the pathological aggregation of tauinto paired helical filaments (PHFs), which are the core constituents ofthe neurofibrillary tangles found in tauopathies. Exemplary sequencesfor the four repeat domains (R1-R4) are provided in SEQ ID NOS: 1-4,respectively. Exemplary coding sequences for the four repeat domains(R1-R4) are provided in SEQ ID NOS: 5-8. An exemplary sequence for theTau four-repeat domain is provided in SEQ ID NO: 9. An exemplary codingsequence for the Tau four-repeat domain is provided in SEQ ID NO: 10. Anexemplary sequence for the Tau four-repeat domain with the P301Smutation is provided in SEQ ID NO: 11. An exemplary coding sequence forthe Tau four-repeat domain with the P301S mutation is provided in SEQ IDNO: 12.

The tau repeat domain used in the Cas/tau biosensor cells or the SAM/taubiosensor cells can comprise the tau microtubule binding domain (MBD).The tau repeat domain used in the Cas/tau biosensor cells or the SAM/taubiosensor cells can comprise one or more or all of the four repeatdomains (R1-R4). For example, the tau repeat domain can comprise,consist essentially of, or consist of one or more or all of SEQ ID NOS:1, 2, 3, and 4, or sequences at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOS: 1, 2, 3, and 4. Inone specific example, the tau repeat domain is the tau four-repeatdomain (R1-R4) found in several tau isoforms. For example, the taurepeat domain can comprise, consist essentially of, or consist of SEQ IDNO: 9 or SEQ ID NO: 11 or a sequence at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9 or SEQ ID NO:11. In one specific example, the nucleic acid encoding the tau repeatdomain can comprise, consist essentially of, or consist of SEQ ID NO: 12or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 12, optionally wherein the nucleicacid encodes a protein comprising, consisting essentially of, orconsisting of SEQ ID NO: 11. In another specific example, the nucleicacid encoding the second tau repeat domain linked to the second reportercan comprise, consist essentially of, or consist of SEQ ID NO: 10 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 10, optionally wherein the nucleic acidencodes a protein comprising, consisting essentially of, or consistingof SEQ ID NO: 9. The first and second tau repeat domains in the cellsdisclosed herein can be the same, similar, or different.

One or both of the first tau repeat domain linked to the first reporterand the second tau repeat domain linked to the second reporter can bestably expressed in the cells. For example, nucleic acids encoding oneor both of the first tau repeat domain linked to the first reporter andthe second tau repeat domain linked to the second reporter can begenomically integrated in the population of cells and operably linked topromoters active in the cell.

The tau repeat domains used in the cells disclosed herein can alsocomprise a tau pathogenic mutation, such as a pro-aggregation mutation.Such a mutation can be, for example, a mutation that is associated with(e.g., segregates with) or causes a tauopathy. As one example, themutation can be an aggregation-sensitizing mutation that sensitizes tauto seeding but does not result in tau readily aggregating on its own.For example, the mutation can be the disease-associated P301S mutation.By P301S mutation is meant the human tau P301S mutation or acorresponding mutation in another tau protein when optimally alignedwith the human tau protein. The P301S mutation in tau exhibits highsensitivity to seeding, but it does not readily aggregate on its own.Thus, although at baseline tau reporter proteins comprising the P301Smutation exist in a stable, soluble form within the cell, exposure toexogenous tau seeds leads to tau reporter protein aggregation. Other taumutations include, for example, K280del, P301L, V337M, P301L/V337M, andK280del/I227P/I308P.

The first tau repeat domain can be linked to the first reporter and thesecond tau repeat domain can be linked to the second reporter by anymeans. For example, the reporter can be fused to the tau repeat domain(e.g., as part of a fusion protein).

The first reporter and second reporter can be and pair of uniquereporters that can act together as an intracellular biosensor thatproduces a detectable signal when the first and second proteins areaggregated. As one example, the reporters can be fluorescent proteins,and fluorescence resonance energy transfer (FRET) can be used to measureprotein aggregation. Specifically, the first and second reporters can bea FRET pair. Examples of FRET pairs (donor and acceptor fluorophores)are well known. See, e.g., Bajar et al. (2016) Sensors (Basel)16(9):1488, herein incorporated by reference in its entirety for allpurposes. Typical fluorescence microscopy techniques rely upon theabsorption by a fluorophore of light at one wavelength (excitation),followed by the subsequent emission of secondary fluorescence at alonger wavelength. The mechanism of fluorescence resonance energytransfer involves a donor fluorophore in an excited electronic state,which may transfer its excitation energy to a nearby acceptorchromophore in a non-radiative fashion through long-range dipole-dipoleinteractions. For example, the FRET energy donor may be the firstreporter, and the FRET energy acceptor may be the second reporter.Alternatively, the FRET energy donor may be the second reporter, and theFRET energy acceptor may be the first reporter. In a specific example,the first and second reporters are CFP and YFP. Exemplary protein andcoding sequences for CFP are provided, e.g., in SEQ ID NOS: 13 and 14,respectively. Exemplary protein and coding sequences for YFP areprovided, e.g., in SEQ ID NOS: 15 and 16, respectively. As a specificexample, the CFP can comprise, consist essentially of, or consist of SEQID NO: 13 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. As another specificexample, the YFP can comprise, consist essentially of, or consist of SEQID NO: 15 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to SEQ ID NO: 15.

As another example, a protein fragment complementation strategy can beused to detect aggregation. For example, a split-luciferase can be usedto produce bioluminescence from a substrate, and the first and secondreporters can be amino- (NLuc) and carboxy- (CLuc) terminal fragments ofthe luciferase. Examples of luciferase include Renilla, firefly, clickbeetle, and Metridia luciferase.

In one non-limiting example, the biosensor cells disclosed hereincontain two transgenes stably expressing disease-associated tau proteinvariants fused to the fluorescent protein CFP or the fluorescent proteinYFP, respectively (tau^(4RD)-CFP/tau^(4RD)-YFP (TCY)), wherein the taufour repeat domain (4RD) comprises the P301S pathogenic mutation. Inthese biosensor lines, tau-CFP/tau-YFP protein aggregation produces aFRET signal, the result of a transfer of fluorescent energy from donorCFP to acceptor YFP. FRET-positive cells, which contain tau aggregates,can be sorted and isolated by flow cytometry. At baseline, unstimulatedcells express the reporters in a stable, soluble state with minimal FRETsignal. Upon stimulation (e.g., liposome transfection of seedparticles), the reporter proteins form aggregates, producing a FRETsignal.

The Cas/tau biosensor cells disclosed herein can be aggregation-positive(Agg[+]) cells in which the tau repeat domain stably presents in anaggregated state, meaning that the tau repeat domain aggregates stablypersist in all cells with growth and multiple passages over time.Alternatively, the Cas/tau biosensor cells disclosed herein can beaggregation-negative (Agg[−]).

2. Cas Proteins and Chimeric Cas Proteins

The Cas/tau biosensor cells disclosed herein also comprise nucleic acids(DNA or RNA) encoding Cas proteins. Optionally, the Cas protein isstably expressed. Optionally, the cells comprise a genomicallyintegrated Cas coding sequence. Likewise, the SAM/tau biosensor cellsdisclosed herein also comprise nucleic acids (DNA or RNA) encodingchimeric Cas proteins comprising a nuclease-inactive Cas protein fusedto one or more transcriptional activation domains (e.g., VP64).Optionally, the chimeric Cas protein is stably expressed. Optionally,the cells comprise a genomically integrated chimeric Cas codingsequence.

Cas proteins are part of Clustered Regularly Interspersed ShortPalindromic Repeats (CRISPR)/CRISPR-associated (Cas) systems. CRISPR/Cassystems include transcripts and other elements involved in theexpression of, or directing the activity of, Cas genes. A CRISPR/Cassystem can be, for example, a type I, a type II, a type III system, or atype V system (e.g., subtype V-A or subtype V-B). The methods andcompositions disclosed herein can employ CRISPR/Cas systems by utilizingCRISPR complexes (comprising a guide RNA (gRNA) complexed with a Casprotein) for site-directed binding or cleavage of nucleic acids.

CRISPR/Cas systems used in the compositions and methods disclosed hereincan be non-naturally occurring. A “non-naturally occurring” systemincludes anything indicating the involvement of the hand of man, such asone or more components of the system being altered or mutated from theirnaturally occurring state, being at least substantially free from atleast one other component with which they are naturally associated innature, or being associated with at least one other component with whichthey are not naturally associated. For example, some CRISPR/Cas systemsemploy non-naturally occurring CRISPR complexes comprising a gRNA and aCas protein that do not naturally occur together, employ a Cas proteinthat does not occur naturally, or employ a gRNA that does not occurnaturally.

Cas proteins generally comprise at least one RNA recognition or bindingdomain that can interact with guide RNAs. Cas proteins can also comprisenuclease domains (e.g., DNase domains or RNase domains), DNA-bindingdomains, helicase domains, protein-protein interaction domains,dimerization domains, and other domains. Some such domains (e.g., DNasedomains) can be from a native Cas protein. Other such domains can beadded to make a modified Cas protein. A nuclease domain possessescatalytic activity for nucleic acid cleavage, which includes thebreakage of the covalent bonds of a nucleic acid molecule. Cleavage canproduce blunt ends or staggered ends, and it can be single-stranded ordouble-stranded. For example, a wild type Cas9 protein will typicallycreate a blunt cleavage product. Alternatively, a wild type Cpf1 protein(e.g., FnCpf1) can result in a cleavage product with a 5-nucleotide 5′overhang, with the cleavage occurring after the 18th base pair from thePAM sequence on the non-targeted strand and after the 23rd base on thetargeted strand. A Cas protein can have full cleavage activity to createa double-strand break at a target genomic locus (e.g., a double-strandbreak with blunt ends), or it can be a nickase that creates asingle-strand break at a target genomic locus.

Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5,Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c,Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3,Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5,Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1,Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1,Csf2, Csf3, Csf4, and Cu1966, and homologs or modified versions thereof.

An exemplary Cas protein is a Cas9 protein or a protein derived from aCas9 protein. Cas9 proteins are from a type II CRISPR/Cas system andtypically share four key motifs with a conserved architecture. Motifs 1,2, and 4 are RuvC-like motifs, and motif 3 is an HNH motif. ExemplaryCas9 proteins are from Streptococcus pyogenes, Streptococcusthermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsisdassonvillei, Streptomyces pristinaespiralis, Streptomycesviridochromogenes, Streptomyces viridochromogenes, Streptosporangiumroseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius,Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacteriumsibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius,Microscilla marina, Burkholderiales bacterium, Polaromonasnaphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothecesp., Microcystis aeruginosa, Synechococcus sp., Acetohalobiumarabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, CandidatusDesulforudis, Clostridium botulinum, Clostridium difficile, Finegoldiamagna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum,Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatiumvinosum, Marinobacter sp Nitrosococcus halophilus, Nitrosococcuswatsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer,Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena,Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp.,Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotogamobilis, Thermosipho africanus, Acaryochloris marina, Neisseriameningitidis, or Campylobacter jejuni. Additional examples of the Cas9family members are described in WO 2014/131833, herein incorporated byreference in its entirety for all purposes. Cas9 from S. pyogenes(SpCas9) (assigned SwissProt accession number Q99ZW2) is an exemplaryCas9 protein. Exemplary SpCas9 protein and coding sequence are set forthin SEQ ID NOS: 21 and 22, respectively. Cas9 from S. aureus (SaCas9)(assigned UniProt accession number J7RUA5) is another exemplary Cas9protein. Cas9 from Campylobacter jejuni (CjCas9) (assigned UniProtaccession number Q0P897) is another exemplary Cas9 protein. See, e.g.,Kim et al. (2017) Nat. Comm. 8:14500, herein incorporated by referencein its entirety for all purposes. SaCas9 is smaller than SpCas9, andCjCas9 is smaller than both SaCas9 and SpCas9. Cas9 from Neisseriameningitidis (Nme2Cas9) is another exemplary Cas9 protein. See, e.g.,Edraki et al. (2019) Mol. Cell 73(4):714-726, herein incorporated byreference in its entirety for all purposes. Cas9 proteins fromStreptococcus thermophilus (e.g., Streptococcus thermophilus LMD-9 Cas9encoded by the CRISPR1 locus (St1Cas9) or Streptococcus thermophilusCas9 from the CRISPR3 locus (St3Cas9)) are other exemplary Cas9proteins. Cas9 from Francisella novicida (FnCas9) or the RHA Francisellanovicida Cas9 variant that recognizes an alternative PAM(E1369R/E1449H/R1556A substitutions) are other exemplary Cas9 proteins.These and other exemplary Cas9 proteins are reviewed, e.g., inCebrian-Serrano and Davies (2017) Mamm. Genome 28(7):247-261, hereinincorporated by reference in its entirety for all purposes.

As one example, the Cas protein can be a Cas9 protein. For example, theCas9 protein can be a Streptococcus pyogenes Cas9 protein. As onespecific example, the Cas protein can comprise, consist essentially of,or consist of SEQ ID NO: 21 or a sequence at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21. Asanother specific example, a chimeric Cas protein comprising anuclease-inactive Cas protein and one or more transcriptional activationdomains can comprise, consist essentially of, or consist of SEQ ID NO:36 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 36.

Another example of a Cas protein is a Cpf1 (CRISPR from Prevotella andFrancisella 1) protein. Cpf1 is a large protein (about 1300 amino acids)that contains a RuvC-like nuclease domain homologous to thecorresponding domain of Cas9 along with a counterpart to thecharacteristic arginine-rich cluster of Cas9. However, Cpf1 lacks theHNH nuclease domain that is present in Cas9 proteins, and the RuvC-likedomain is contiguous in the Cpf1 sequence, in contrast to Cas9 where itcontains long inserts including the HNH domain. See, e.g., Zetsche etal. (2015) Cell 163(3):759-771, herein incorporated by reference in itsentirety for all purposes. Exemplary Cpf1 proteins are from Francisellatularensis 1, Francisella tularensis subsp. novicida, Prevotellaalbensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrioproteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10,Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC,Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, CandidatusMethanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237,Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonascrevioricanis 3, Prevotella disiens, and Porphyromonas macacae. Cpf1from Francisella novicida U112 (FnCpf1; assigned UniProt accessionnumber A0Q7Q2) is an exemplary Cpf1 protein.

Cas proteins can be wild type proteins (i.e., those that occur innature), modified Cas proteins (i.e., Cas protein variants), orfragments of wild type or modified Cas proteins. Cas proteins can alsobe active variants or fragments with respect to catalytic activity ofwild type or modified Cas proteins. Active variants or fragments withrespect to catalytic activity can comprise at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thewild type or modified Cas protein or a portion thereof, wherein theactive variants retain the ability to cut at a desired cleavage site andhence retain double-strand-break-inducing activity. Assays fordouble-strand-break-inducing activity are known and generally measurethe overall activity and specificity of the Cas protein on DNAsubstrates containing the cleavage site.

One example of a modified Cas protein is the modified SpCas9-HF1protein, which is a high-fidelity variant of Streptococcus pyogenes Cas9harboring alterations (N497A/R661A/Q695A/Q926A) designed to reducenon-specific DNA contacts. See, e.g., Kleinstiver et al. (2016) Nature529(7587):490-495, herein incorporated by reference in its entirety forall purposes. Another example of a modified Cas protein is the modifiedeSpCas9 variant (K848A/K1003A/R1060A) designed to reduce off-targeteffects. See, e.g., Slaymaker et al. (2016) Science 351(6268):84-88,herein incorporated by reference in its entirety for all purposes. OtherSpCas9 variants include K855A and K810A/K1003A/R1060A. These and othermodified Cas proteins are reviewed, e.g., in Cebrian-Serrano and Davies(2017) Mamm. Genome 28(7):247-261, herein incorporated by reference inits entirety for all purposes. Another example of a modified Cas9protein is xCas9, which is a SpCas9 variant that can recognize anexpanded range of PAM sequences. See, e.g., Hu et al. (2018) Nature556:57-63, herein incorporated by reference in its entirety for allpurposes.

Cas proteins can be modified to increase or decrease one or more ofnucleic acid binding affinity, nucleic acid binding specificity, andenzymatic activity. Cas proteins can also be modified to change anyother activity or property of the protein, such as stability. Forexample, a Cas protein can be truncated to remove domains that are notessential for the function of the protein or to optimize (e.g., enhanceor reduce) the activity of or a property of the Cas protein. As anotherexample, one or more nuclease domains of the Cas protein can bemodified, deleted, or inactivated (e.g., for use in the SAM/taubiosensor cells comprising a nuclease-inactive Cas protein).

Cas proteins can comprise at least one nuclease domain, such as a DNasedomain. For example, a wild type Cpf1 protein generally comprises aRuvC-like domain that cleaves both strands of target DNA, perhaps in adimeric configuration. Cas proteins can also comprise at least twonuclease domains, such as DNase domains. For example, a wild type Cas9protein generally comprises a RuvC-like nuclease domain and an HNH-likenuclease domain. The RuvC and HNH domains can each cut a differentstrand of double-stranded DNA to make a double-stranded break in theDNA. See, e.g., Jinek et al. (2012) Science 337:816-821, hereinincorporated by reference in its entirety for all purposes.

One or more or all of the nuclease domains can be deleted or mutated sothat they are no longer functional or have reduced nuclease activity.For example, if one of the nuclease domains is deleted or mutated in aCas9 protein, the resulting Cas9 protein can be referred to as a nickaseand can generate a single-strand break within a double-stranded targetDNA but not a double-strand break (i.e., it can cleave the complementarystrand or the non-complementary strand, but not both). If both of thenuclease domains are deleted or mutated, the resulting Cas protein(e.g., Cas9) will have a reduced ability to cleave both strands of adouble-stranded DNA (e.g., a nuclease-null or nuclease-inactive Casprotein, or a catalytically dead Cas protein (dCas)). An example of amutation that converts Cas9 into a nickase is a D10A (aspartate toalanine at position 10 of Cas9) mutation in the RuvC domain of Cas9 fromS. pyogenes. Likewise, H939A (histidine to alanine at amino acidposition 839), H840A (histidine to alanine at amino acid position 840),or N863A (asparagine to alanine at amino acid position N863) in the HNHdomain of Cas9 from S. pyogenes can convert the Cas9 into a nickase.Other examples of mutations that convert Cas9 into a nickase include thecorresponding mutations to Cas9 from S. thermophilus. See, e.g.,Sapranauskas et al. (2011) Nucleic Acids Res. 39(21):9275-9282 and WO2013/141680, each of which is herein incorporated by reference in itsentirety for all purposes. Such mutations can be generated using methodssuch as site-directed mutagenesis, PCR-mediated mutagenesis, or totalgene synthesis. Examples of other mutations creating nickases can befound, for example, in WO 2013/176772 and WO 2013/142578, each of whichis herein incorporated by reference in its entirety for all purposes. Ifall of the nuclease domains are deleted or mutated in a Cas protein(e.g., both of the nuclease domains are deleted or mutated in a Cas9protein), the resulting Cas protein (e.g., Cas9) will have a reducedability to cleave both strands of a double-stranded DNA (e.g., anuclease-null or nuclease-inactive Cas protein). One specific example isa D10A/H840A S. pyogenes Cas9 double mutant or a corresponding doublemutant in a Cas9 from another species when optimally aligned with S.pyogenes Cas9. Another specific example is a D10A/N863A S. pyogenes Cas9double mutant or a corresponding double mutant in a Cas9 from anotherspecies when optimally aligned with S. pyogenes Cas9.

Examples of inactivating mutations in the catalytic domains of xCas9 arethe same as those described above for SpCas9. Examples of inactivatingmutations in the catalytic domains of Staphylococcus aureus Cas9proteins are also known. For example, the Staphyloccocus aureus Cas9enzyme (SaCas9) may comprise a substitution at position N580 (e.g.,N580A substitution) and a substitution at position D10 (e.g., D10Asubstitution) to generate a nuclease-inactive Cas protein. See, e.g., WO2016/106236, herein incorporated by reference in its entirety for allpurposes. Examples of inactivating mutations in the catalytic domains ofNme2Cas9 are also known (e.g., combination of D16A and H588A). Examplesof inactivating mutations in the catalytic domains of St1Cas9 are alsoknown (e.g., combination of D9A, D598A, H599A, and N622A). Examples ofinactivating mutations in the catalytic domains of St3Cas9 are alsoknown (e.g., combination of D10A and N870A). Examples of inactivatingmutations in the catalytic domains of CjCas9 are also known (e.g.,combination of D8A and H559A). Examples of inactivating mutations in thecatalytic domains of FnCas9 and RHA FnCas9 are also known (e.g., N995A).

Examples of inactivating mutations in the catalytic domains of Cpf1proteins are also known. With reference to Cpf1 proteins fromFrancisella novicida U112 (FnCpf1), Acidaminococcus sp. BV3L6 (AsCpf1),Lachnospiraceae bacterium ND2006 (LbCpf1), and Moraxella bovoculi 237(MbCpf1 Cpf1), such mutations can include mutations at positions 908,993, or 1263 of AsCpf1 or corresponding positions in Cpf1 orthologs, orpositions 832, 925, 947, or 1180 of LbCpf1 or corresponding positions inCpf1 orthologs. Such mutations can include, for example one or more ofmutations D908A, E993A, and D1263A of AsCpf1 or corresponding mutationsin Cpf1 orthologs, or D832A, E925A, D947A, and D1180A of LbCpf1 orcorresponding mutations in Cpf1 orthologs. See, e.g., US 2016/0208243,herein incorporated by reference in its entirety for all purposes.

Cas proteins can also be operably linked to heterologous polypeptides asfusion proteins. For example, a Cas protein can be fused to a cleavagedomain, an epigenetic modification domain, a transcriptional activationdomain, or a transcriptional repressor domain. See WO 2014/089290,herein incorporated by reference in its entirety for all purposes. Forexample, Cas proteins can be operably linked or fused to atranscriptional activation domain for use in the SAM/tau biosensorcells. Examples of transcriptional activation domains include a herpessimplex virus VP16 activation domain, VP64 (which is a tetramericderivative of VP16), a NFκB p65 activation domain, p53 activationdomains 1 and 2, a CREB (cAMP response element binding protein)activation domain, an E2A activation domain, and an NFAT (nuclear factorof activated T-cells) activation domain. Other examples includeactivation domains from Octl, Oct-2A, SP1, AP-2, CTF1, P300, CBP, PCAF,SRC1, PvALF, ERF-2, OsGAI, HALF-1, Cl, AP1, ARF-5, ARF-6, ARF-7, ARF-8,CPRF1, CPRF4, MYC-RP/GP, TRAB1PC4, and HSF1. See, e.g., US 2016/0237456,EP3045537, and WO 2011/146121, each of which is incorporated byreference in its entirety for all purposes. In some cases, atranscriptional activation system can be used comprising a dCas9-VP64fusion protein paired with MS2-p65-HSF1. Guide RNAs in such systems canbe designed with aptamer sequences appended to sgRNA tetraloop andstem-loop 2 designed to bind dimerized MS2 bacteriophage coat proteins.See, e.g., Konermann et al. (2015) Nature 517(7536):583-588, hereinincorporated by reference in its entirety for all purposes. Examples oftranscriptional repressor domains include inducible cAMP early repressor(ICER) domains, Kruppel-associated box A (KRAB-A) repressor domains, YY1glycine rich repressor domains, Sp1-like repressors, E(spl) repressors,IκB repressor, and MeCP2. Other examples include transcriptionalrepressor domains from A/B, KOX, TGF-beta-inducible early gene (TIEG),v-erbA, SID, SID4X, MBD2, MBD3, DNMT1, DNMG3A, DNMT3B, Rb, ROM2, See,e.g., EP3045537 and WO 2011/146121, each of which is incorporated byreference in its entirety for all purposes. Cas proteins can also befused to a heterologous polypeptide providing increased or decreasedstability. The fused domain or heterologous polypeptide can be locatedat the N-terminus, the C-terminus, or internally within the Cas protein.

Cas proteins can also be operably linked to heterologous polypeptides asfusion proteins. As one example, a Cas protein can be fused to one ormore heterologous polypeptides that provide for subcellularlocalization. Such heterologous polypeptides can include, for example,one or more nuclear localization signals (NLS) such as the monopartiteSV40 NLS and/or a bipartite alpha-importin NLS for targeting to thenucleus, a mitochondrial localization signal for targeting to themitochondria, an ER retention signal, and the like. See, e.g., Lange etal. (2007) J Biol. Chem. 282:5101-5105, herein incorporated by referencein its entirety for all purposes. Such subcellular localization signalscan be located at the N-terminus, the C-terminus, or anywhere within theCas protein. An NLS can comprise a stretch of basic amino acids, and canbe a monopartite sequence or a bipartite sequence. Optionally, a Casprotein can comprise two or more NLSs, including an NLS (e.g., analpha-importin NLS or a monopartite NLS) at the N-terminus and an NLS(e.g., an SV40 NLS or a bipartite NLS) at the C-terminus. A Cas proteincan also comprise two or more NLSs at the N-terminus and/or two or moreNLSs at the C-terminus.

Cas proteins can also be operably linked to a cell-penetrating domain orprotein transduction domain. For example, the cell-penetrating domaincan be derived from the HIV-1 TAT protein, the TLM cell-penetratingmotif from human hepatitis B virus, MPG, Pep-1, VP22, a cell penetratingpeptide from Herpes simplex virus, or a polyarginine peptide sequence.See, e.g., WO 2014/089290 and WO 2013/176772, each of which is hereinincorporated by reference in its entirety for all purposes. Thecell-penetrating domain can be located at the N-terminus, theC-terminus, or anywhere within the Cas protein.

Cas proteins can also be operably linked to a heterologous polypeptidefor ease of tracking or purification, such as a fluorescent protein, apurification tag, or an epitope tag. Examples of fluorescent proteinsinclude green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP,eGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP,ZsGreenl), yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus,YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., eBFP, eBFP2,Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescentproteins (e.g., eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), redfluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer,mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem,HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), orangefluorescent proteins (e.g., mOrange, mKO, Kusabira-Orange, MonomericKusabira-Orange, mTangerine, tdTomato), and any other suitablefluorescent protein. Examples of tags include glutathione-S-transferase(GST), chitin binding protein (CBP), maltose binding protein,thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag,myc, AcV5, AU1, AUS, E, ECS, E2, FLAG, hemagglutinin (HA), nus, Softag1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, 51, T7, V5, VSV-G,histidine (His), biotin carboxyl carrier protein (BCCP), and calmodulin.

Cas proteins can be provided in any form. For example, a Cas protein canbe provided in the form of a protein. For example, a Cas protein can beprovided as a Cas protein complexed with a gRNA. Alternatively, a Casprotein can be provided in the form of a nucleic acid encoding the Casprotein, such as an RNA (e.g., messenger RNA (mRNA)) or DNA. Optionally,the nucleic acid encoding the Cas protein can be codon optimized forefficient translation into protein in a particular cell or organism. Forexample, the nucleic acid encoding the Cas protein can be modified tosubstitute codons having a higher frequency of usage in a bacterialcell, a yeast cell, a human cell, a non-human cell, a mammalian cell, arodent cell, a mouse cell, a rat cell, or any other host cell ofinterest, as compared to the naturally occurring polynucleotidesequence. For example, the nucleic acid encoding the Cas protein can becodon optimized for expression in a human cell. When a nucleic acidencoding the Cas protein is introduced into the cell, the Cas proteincan be transiently, conditionally, or constitutively expressed in thecell.

Cas proteins provided as mRNAs can be modified for improved stabilityand/or immunogenicity properties. The modifications may be made to oneor more nucleosides within the mRNA. Examples of chemical modificationsto mRNA nucleobases include pseudouridine, 1-methyl-pseudouridine, and5-methyl-cytidine. For example, capped and polyadenylated Cas mRNAcontaining N1-methyl pseudouridine can be used. Likewise, Cas mRNAs canbe modified by depletion of uridine using synonymous codons.

Nucleic acids encoding Cas proteins can be stably integrated in thegenome of a cell and operably linked to a promoter active in the cell.In one specific example, the nucleic acid encoding the Cas protein cancomprise, consist essentially of, or consist of SEQ ID NO: 22 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 22, optionally wherein the nucleic acidencodes a protein comprising, consisting essentially of, or consistingof SEQ ID NO: 21. In another specific example, the nucleic acid encodinga chimeric Cas protein comprising a nuclease-inactive Cas protein andone or more transcriptional activation domains can comprise, consistessentially of, or consist of SEQ ID NO: 38 or a sequence at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO: 38, optionally wherein the nucleic acid encodes a proteincomprising, consisting essentially of, or consisting of SEQ ID NO: 36.Alternatively, nucleic acids encoding Cas proteins can be operablylinked to a promoter in an expression construct. Expression constructsinclude any nucleic acid constructs capable of directing expression of agene or other nucleic acid sequence of interest (e.g., a Cas gene) andwhich can transfer such a nucleic acid sequence of interest to a targetcell. Promoters that can be used in an expression construct includepromoters active, for example, in one or more of a eukaryotic cell, ahuman cell, a non-human cell, a mammalian cell, a non-human mammaliancell, a rodent cell, a mouse cell, a rat cell, a pluripotent cell, anembryonic stem (ES) cell, an adult stem cell, a developmentallyrestricted progenitor cell, an induced pluripotent stem (iPS) cell, or aone-cell stage embryo. Such promoters can be, for example, conditionalpromoters, inducible promoters, constitutive promoters, ortissue-specific promoters.

3. Chimeric Adaptor Proteins

The SAM/tau biosensor cells disclosed herein can comprise not onlynucleic acids (DNA or RNA) encoding a chimeric Cas protein comprising anuclease-inactive Cas protein fused to one or more transcriptionalactivation domains (e.g., VP64) but optionally also nucleic acids (DNAor RNA) encoding a chimeric adaptor protein comprising an adaptorprotein (e.g., MS2 coat protein (MCP)) fused to one or moretranscriptional activation domains (e.g., fused to p65 and HSF1).Optionally, the chimeric Cas protein and/or the chimeric adaptor proteinis stably expressed. Optionally, the cells comprise a genomicallyintegrated chimeric Cas protein coding sequence and/or a genomicallyintegrated chimeric adaptor protein coding sequence.

Such chimeric adaptor proteins comprise: (a) an adaptor (i.e., adaptordomain or adaptor protein) that specifically binds to an adaptor-bindingelement within a guide RNA; and (b) one or more heterologoustranscriptional activation domains. For example, such fusion proteinscan comprise 1, 2, 3, 4, 5, or more transcriptional activation domains(e.g., two or more heterologous transcriptional activation domains orthree or more heterologous transcriptional activation domains). In oneexample, such chimeric adaptor proteins can comprise: (a) an adaptor(i.e., an adaptor domain or adaptor protein) that specifically binds toan adaptor-binding element in a guide RNA; and (b) two or moretranscriptional activation domains. For example, the chimeric adaptorprotein can comprise: (a) an MS2 coat protein adaptor that specificallybinds to one or more MS2 aptamers in a guide RNA (e.g., two MS2 aptamersin separate locations in a guide RNA); and (b) one or more (e.g., two ormore transcriptional activation domains). For example, the twotranscriptional activation domains can be p65 and HSF1 transcriptionalactivation domains or functional fragments or variants thereof. However,chimeric adaptor proteins in which the transcriptional activationdomains comprise other transcriptional activation domains or functionalfragments or variants thereof are also provided.

The one or more transcriptional activation domains can be fused directlyto the adaptor. Alternatively, the one or more transcriptionalactivation domains can be linked to the adaptor via a linker or acombination of linkers or via one or more additional domains. Likewise,if two or more transcriptional activation domains are present, they canbe fused directly to each other or can be linked to each other via alinker or a combination of linkers or via one or more additionaldomains. Linkers that can be used in these fusion proteins can includeany sequence that does not interfere with the function of the fusionproteins. Exemplary linkers are short (e.g., 2-20 amino acids) and aretypically flexible (e.g., comprising amino acids with a high degree offreedom such as glycine, alanine, and serine).

The one or more transcriptional activation domains and the adaptor canbe in any order within the chimeric adaptor protein. As one option, theone or more transcriptional activation domains can be C-terminal to theadaptor and the adaptor can be N-terminal to the one or moretranscriptional activation domains. For example, the one or moretranscriptional activation domains can be at the C-terminus of thechimeric adaptor protein, and the adaptor can be at the N-terminus ofthe chimeric adaptor protein. However, the one or more transcriptionalactivation domains can be C-terminal to the adaptor without being at theC-terminus of the chimeric adaptor protein (e.g., if a nuclearlocalization signal is at the C-terminus of the chimeric adaptorprotein). Likewise, the adaptor can be N-terminal to the one or moretranscriptional activation domains without being at the N-terminus ofthe chimeric adaptor protein (e.g., if a nuclear localization signal isat the N-terminus of the chimeric adaptor protein). As another option,the one or more transcriptional activation domains can be N-terminal tothe adaptor and the adaptor can be C-terminal to the one or moretranscriptional activation domains. For example, the one or moretranscriptional activation domains can be at the N-terminus of thechimeric adaptor protein, and the adaptor can be at the C-terminus ofthe chimeric adaptor protein. As yet another option, if the chimericadaptor protein comprises two or more transcriptional activationdomains, the two or more transcriptional activation domains can flankthe adaptor.

Chimeric adaptor proteins can also be operably linked or fused toadditional heterologous polypeptides. The fused or linked heterologouspolypeptide can be located at the N-terminus, the C-terminus, oranywhere internally within the chimeric adaptor protein. For example, achimeric adaptor protein can further comprise a nuclear localizationsignal. A specific example of such a protein comprises an MS2 coatprotein (adaptor) linked (either directly or via an NLS) to a p65transcriptional activation domain C-terminal to the MS2 coat protein(MCP), and HSF1 transcriptional activation domain C-terminal to the p65transcriptional activation domain. Such a protein can comprise fromN-terminus to C-terminus: an MCP; a nuclear localization signal; a p65transcriptional activation domain; and an HSF1 transcriptionalactivation domain. For example, a chimeric adaptor protein can comprise,consist essentially of, or consist of an amino acid sequence at least85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto the MCP-p65-HSF1 chimeric adaptor protein sequence set forth in SEQID NO: 37. Likewise, a nucleic acid encoding a chimeric adaptor proteincan comprise, consist essentially of, or consist of a sequence at least85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto the MCP-p65-HSF1 chimeric adaptor protein coding sequence set forthin SEQ ID NO: 39

Adaptors (i.e., adaptor domains or adaptor proteins) arenucleic-acid-binding domains (e.g., DNA-binding domains and/orRNA-binding domains) that specifically recognize and bind to distinctsequences (e.g., bind to distinct DNA and/or RNA sequences such asaptamers in a sequence-specific manner). Aptamers include nucleic acidsthat, through their ability to adopt a specific three-dimensionalconformation, can bind to a target molecule with high affinity andspecificity. Such adaptors can bind, for example, to a specific RNAsequence and secondary structure. These sequences (i.e., adaptor-bindingelements) can be engineered into a guide RNA. For example, an MS2aptamer can be engineered into a guide RNA to specifically bind an MS2coat protein (MCP). For example, the adaptor can comprise, consistessentially of, or consist of an amino acid sequence at least 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theMCP sequence set forth in SEQ ID NO: 40. Likewise, a nucleic acidencoding the adaptor can comprise, consist essentially of, or consist ofan amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the MCP coding sequence set forth inSEQ ID NO: 41. Specific examples of adaptors and targets include, forexample, RNA-binding protein/aptamer combinations that exist within thediversity of bacteriophage coat proteins. See, e.g., US 2019-0284572 andWO 2019/183123, each of which is herein incorporated by reference in itsentirety for all purposes.

The chimeric adaptor proteins disclosed herein comprise one or moretranscriptional activation domains. Such transcriptional activationdomains can be naturally occurring transcriptional activation domains,can be functional fragments or functional variants of naturallyoccurring transcriptional activation domains, or can be engineered orsynthetic transcriptional activation domains. Transcriptional activationdomains that can be used include those described, for example, in US2019-0284572 and WO 2019/183123, each of which is herein incorporated byreference in its entirety for all purposes.

4. Cell Types

The Cas/tau biosensor cells disclosed herein can be any type of cell andcan be in vitro, ex vivo, or in vivo. A Cas/tau biosensor cell line orpopulation of cells can be a monoclonal cell line or population ofcells. Likewise, the SAM/tau biosensor cells disclosed herein can be anytype of cell and can be in vitro, ex vivo, or in vivo. A SAM/taubiosensor cell line or population of cells can be a monoclonal cell lineor population of cells. The cell can be from any source. For example,the cell can be a eukaryotic cell, an animal cell, a plant cell, or afungal (e.g., yeast) cell. Such cells can be fish cells or bird cells,or such cells can be mammalian cells, such as human cells, non-humanmammalian cells, rodent cells, mouse cells, or rat cells. Mammalsinclude, for example, humans, non-human primates, monkeys, apes, catsdogs, horses, bulls, deer, bison, sheep, rodents (e.g., mice, rats,hamsters, guinea pigs), livestock (e.g., bovine species such as cows andsteer; ovine species such as sheep and goats; and porcine species suchas pigs and boars). Birds include, for example, chickens, turkeys,ostrich, geese, and ducks. Domesticated animals and agricultural animalsare also included. The term “non-human animal” excludes humans. In aspecific example, the Cas/tau biosensor cells are human cells (e.g.,HEK293T cells). Likewise, in a specific example, the SAM/tau biosensorcells are human cells (e.g., HEK293T cells).

The cell can be, for example, a totipotent cell or a pluripotent cell(e.g., an embryonic stem (ES) cell such as a rodent ES cell, a mouse EScell, or a rat ES cell). Totipotent cells include undifferentiated cellsthat can give rise to any cell type, and pluripotent cells includeundifferentiated cells that possess the ability to develop into morethan one differentiated cell types. Such pluripotent and/or totipotentcells can be, for example, ES cells or ES-like cells, such as an inducedpluripotent stem (iPS) cells. ES cells include embryo-derived totipotentor pluripotent cells that are capable of contributing to any tissue ofthe developing embryo upon introduction into an embryo. ES cells can bederived from the inner cell mass of a blastocyst and are capable ofdifferentiating into cells of any of the three vertebrate germ layers(endoderm, ectoderm, and mesoderm).

The cell can also be a primary somatic cell, or a cell that is not aprimary somatic cell. Somatic cells can include any cell that is not agamete, germ cell, gametocyte, or undifferentiated stem cell. The cellcan also be a primary cell. Primary cells include cells or cultures ofcells that have been isolated directly from an organism, organ, ortissue. Primary cells include cells that are neither transformed norimmortal. They include any cell obtained from an organism, organ, ortissue which was not previously passed in tissue culture or has beenpreviously passed in tissue culture but is incapable of beingindefinitely passed in tissue culture. Such cells can be isolated byconventional techniques and include, for example, somatic cells,hematopoietic cells, endothelial cells, epithelial cells, fibroblasts,mesenchymal cells, keratinocytes, melanocytes, monocytes, mononuclearcells, adipocytes, preadipocytes, neurons, glial cells, hepatocytes,skeletal myoblasts, and smooth muscle cells. For example, primary cellscan be derived from connective tissues, muscle tissues, nervous systemtissues, or epithelial tissues.

Such cells also include would normally not proliferate indefinitely but,due to mutation or alteration, have evaded normal cellular senescenceand instead can keep undergoing division. Such mutations or alterationscan occur naturally or be intentionally induced. Examples ofimmortalized cells include Chinese hamster ovary (CHO) cells, humanembryonic kidney cells (e.g., HEK293T cells), and mouse embryonicfibroblast cells (e.g., 3T3 cells). Numerous types of immortalized cellsare well known. Immortalized or primary cells include cells that aretypically used for culturing or for expressing recombinant genes orproteins.

The cell can also be a differentiated cell, such as a neuronal cell(e.g., a human neuronal cell).

B. Methods of Generating Cas/Tau Biosensor Cells and SAM/Tau BiosensorCells

The Cas/tau biosensor cells disclosed herein can be generated by anyknown means. The first tau repeat domain linked to the first reporter,the second tau repeat domain linked to the second reporter, and the Casprotein can be introduced into the cell in any form (e.g., DNA, RNA, orprotein) by any known means. Likewise, the SAM/tau biosensor cellsdisclosed herein can be generated by any known means. The first taurepeat domain linked to the first reporter, the second tau repeat domainlinked to the second reporter, the chimeric Cas protein, and thechimeric adaptor protein can be introduced into the cell in any form(e.g., DNA, RNA, or protein) by any known means. “Introducing” includespresenting to the cell the nucleic acid or protein in such a manner thatthe sequence gains access to the interior of the cell. The methodsprovided herein do not depend on a particular method for introducing anucleic acid or protein into the cell, only that the nucleic acid orprotein gains access to the interior of a least one cell. Methods forintroducing nucleic acids and proteins into various cell types are knownand include, for example, stable transfection methods, transienttransfection methods, and virus-mediated methods. Optionally, targetingvectors can be used.

Transfection protocols as well as protocols for introducing nucleicacids or proteins into cells may vary. Non-limiting transfection methodsinclude chemical-based transfection methods using liposomes;nanoparticles; calcium phosphate (Graham et al. (1973) Virology 52 (2):456-67, Bacchetti et al. (1977) Proc. Natl. Acad. Sci. USA 74 (4):1590-4, and Kriegler, M (1991). Transfer and Expression: A LaboratoryManual. New York: W. H. Freeman and Company. pp. 96-97); dendrimers; orcationic polymers such as DEAE-dextran or polyethylenimine. Non-chemicalmethods include electroporation, Sono-poration, and opticaltransfection. Particle-based transfection includes the use of a genegun, or magnet-assisted transfection (Bertram (2006) CurrentPharmaceutical Biotechnology 7, 277-28). Viral methods can also be usedfor transfection.

Introduction of nucleic acids or proteins into a cell can also bemediated by electroporation, by intracytoplasmic injection, by viralinfection, by adenovirus, by adeno-associated virus, by lentivirus, byretrovirus, by transfection, by lipid-mediated transfection, or bynucleofection. Nucleofection is an improved electroporation technologythat enables nucleic acid substrates to be delivered not only to thecytoplasm but also through the nuclear membrane and into the nucleus. Inaddition, use of nucleofection in the methods disclosed herein typicallyrequires much fewer cells than regular electroporation (e.g., only about2 million compared with 7 million by regular electroporation). In oneexample, nucleofection is performed using the LONZA® NUCLEOFECTOR™system.

Introduction of nucleic acids or proteins into a cell can also beaccomplished by microinjection. Microinjection of an mRNA is preferablyinto the cytoplasm (e.g., to deliver mRNA directly to the translationmachinery), while microinjection of a protein or a DNA encoding aprotein is preferably into the nucleus. Alternatively, microinjectioncan be carried out by injection into both the nucleus and the cytoplasm:a needle can first be introduced into the nucleus and a first amount canbe injected, and while removing the needle from the cell a second amountcan be injected into the cytoplasm. Methods for carrying outmicroinjection are well known. See, e.g., Nagy et al. (Nagy A,Gertsenstein M, Vintersten K, Behringer R., 2003, Manipulating the MouseEmbryo. Cold Spring Harbor, New York: Cold Spring Harbor LaboratoryPress); Meyer et al. (2010) Proc. Natl. Acad. Sci. USA 107:15022-15026and Meyer et al. (2012) Proc. Natl. Acad. Sci. USA 109:9354-9359.

Other methods for introducing nucleic acid or proteins into a cell caninclude, for example, vector delivery, particle-mediated delivery,exosome-mediated delivery, lipid-nanoparticle-mediated delivery,cell-penetrating-peptide-mediated delivery, orimplantable-device-mediated delivery. Methods of administering nucleicacids or proteins to a subject to modify cells in vivo are disclosedelsewhere herein.

In one example, the first tau repeat domain linked to the firstreporter, the second tau repeat domain linked to the second reporter,and the Cas protein can be introduced via viral transduction such aslentiviral transduction.

Screening for cells comprising the first tau repeat domain linked to thefirst reporter, the second tau repeat domain linked to the secondreporter, and the Cas protein can be performed by any known means.

As one example, reporter genes can be used to screen for cells that havethe Cas protein, the first tau repeat domain linked to the firstreporter, or the second tau repeat domain linked to the second reporter.Exemplary reporter genes include those encoding luciferase,β-galactosidase, green fluorescent protein (GFP), enhanced greenfluorescent protein (eGFP), cyan fluorescent protein (CFP), yellowfluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP),blue fluorescent protein (BFP), enhanced blue fluorescent protein(eBFP), DsRed, ZsGreen, MmGFP, mPlum, mCherry, tdTomato, mStrawberry,J-Red, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, Cerulean,T-Sapphire, and alkaline phosphatase. For example, if the first reporterand the second reporter are fluorescent proteins (e.g., CFP and YFP),cells comprising these reporters can be selected by flow cytometry toselect for dual-positive cells. The dual-positive cells can then becombined to generate a polyclonal line, or monoclonal lines can begenerated from single dual-positive cells.

As another example, selection markers can be used to screen for cellsthat have the Cas protein, the first tau repeat domain linked to thefirst reporter, or the second tau repeat domain linked to the secondreporter. Exemplary selection markers include neomycinphosphotransferase (neo^(r)), hygromycin B phosphotransferase (hyg^(r)),puromycin-N-acetyltransferase (puro^(r)), blasticidin S deaminase(bsr^(r)), xanthine/guanine phosphoribosyl transferase (gpt), or herpessimplex virus thymidine kinase (HSV-k). Another exemplary selectionmarker is bleomycin resistance protein, encoded by the Sh ble gene(Streptoalloteichus hindustanus bleomycin gene), which confers zeocin(phleomycin D1) resistance.

Aggregation-positive (Agg[+]) cells in which the tau repeat domainstably presents in an aggregated state, meaning that the tau repeatdomain aggregates stably persist in all cells with growth and multiplepassages over time, can be generated, for example, by seeding with tauaggregates. For example, naïve aggregation-negative (Agg[−]) Cas/taubiosensor cells disclosed herein can be treated with recombinantfibrillized tau (e.g., recombinant fibrillized tau repeat domain) toseed the aggregation of the tau repeat domain protein stably expressedby these cells. Likewise, naïve aggregation-negative (Agg[−]) SAM/taubiosensor cells disclosed herein can be treated with recombinantfibrillized tau (e.g., recombinant fibrillized tau repeat domain) toseed the aggregation of the tau repeat domain protein stably expressedby these cells. The fibrillized tau repeat domain can be the same as,similar to, or different from the tau repeat domain stably expressed bythe cells. Optionally, the recombinant fibrillized tau can be mixed withlipofectamine reagent. The seeded cells can then be serially diluted toobtain single-cell-derived clones and to identify clonal cell lines inwhich tau repeat domain aggregates stably persist in all cells withgrowth and multiple passages over time.

C. In Vitro Cultures

Also disclosed herein are in vitro cultures or compositions comprisingthe Cas/tau biosensor cells disclosed herein and medium for culturingthose cells. Also disclosed herein are in vitro cultures or compositionscomprising the SAM/tau biosensor cells disclosed herein and medium forculturing those cells. The cells can be Agg[−] cells or Agg[+] cells.

III. Guide RNA Knockout Libraries

The CRISPRn screening methods disclosed herein make use of CRISPR guideRNA (gRNA) knockout libraries such as genome-wide gRNA knockoutlibraries. Cas nucleases such as Cas9 can be programmed to inducedouble-strand breaks at specific genomic loci through gRNAs designed totarget specific target sequences. Because the targeting specificity ofCas proteins is conferred by short gRNAs, pooled genome-scale functionalscreening is possible. Such libraries have several advantages overlibraries such as shRNA libraries, which reduce protein expression bytargeting mRNA. In contrast, gRNA libraries achieve knockout viaframeshift mutations introduced in genomic coding regions of genes.

The CRISPRa screening methods disclosed herein make use of CRISPR guideRNA (gRNA) transcriptional activation libraries such as genome-wide gRNAtranscriptional activation libraries. SAM systems can be programmed toactivate transcription of genes at specific genomic loci through gRNAsdesigned to target specific target sequences. Because the targetingspecificity of Cas proteins is conferred by short gRNAs, pooledgenome-scale functional screening is possible.

The gRNAs in a library can target any number of genes. For example, thegRNAs can target about 50 or more genes, about 100 or more genes, about200 or more genes, about 300 or more genes, about 400 or more genes,about 500 or more genes, about 1000 or more genes, about 2000 or moregenes, about 3000 or more genes, about 4000 or more genes, about 5000 ormore genes, about 10000 or more genes, or about 20000 or more genes. Insome libraries, the gRNAs can be selected to target genes in aparticular signaling pathway. Some libraries are genome-wide libraries.

The genome-wide libraries include one or more gRNAs (e.g., sgRNAs)targeting each gene in the genome. The genome being targeted can anytype of genome. For example, the genome can be a eukaryotic genome, amammalian genome, a non-human mammalian genome, a rodent genome, a mousegenome, a rat genome, or a human genome. In one example, the targetedgenome is a human genome.

The gRNAs can target any number of sequences within each individualtargeted gene. In some libraries, a plurality of target sequences aretargeted on average in each of the targeted plurality of genes. Forexample, about 2 to about 10, about 2 to about 9, about 2 to about 8,about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 toabout 4, or about 2 to about 3 unique target sequences can be targetedon average in each of the targeted plurality of genes. For example, atleast about 2, at least about 3, at least about 4, at least about 5, orat least about 6 unique target sequences can be targeted on average ineach of the targeted plurality of genes. As a specific example, about 6target sequences can be targeted on average in each of the targetedplurality of genes. As another specific example, about 3 to about 6 orabout 4 to about 6 target sequences are targeted on average in each ofthe targeted plurality of genes.

For example, the libraries can target genes with an average coverage ofabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, or about 10 gRNAs per gene. In a specific example a library cantarget genes with an average coverage of about 3-4 gRNAs per gene orabout 6 gRNAs per gene.

The gRNAs can target any desired location in the target genes. TheCRISPRn gRNAs can be designed to target coding regions of genes so thatcleavage by the corresponding Cas protein will result in frameshiftinsertion/deletion (indel) mutations that result in a loss-of-functionallele. More specifically, frameshift mutations can be achieved throughtargeted DNA double strand breaks and subsequent mutagenic repair viathe non-homologous end joining (NHEJ) pathway, which produces indels atthe site of break. The indel being introduced into the DSB is random,with some indels leading to frameshift mutations that cause prematuretermination of the gene.

In some CRISPRn libraries, each gRNA targets a constitutive exon ifpossible. In some CRISPRn libraries, each gRNA targets a 5′ constitutiveexon if possible. In some methods, each gRNA targets a first exon, asecond exon, or a third exon (from the 5′ end of the gene) if possible.

As one example, the gRNAs in the CRISPRn library can target constitutiveexons. Constitutive exons are exons that are consistently conservedafter splicing. Exons expressed across all tissues can be consideredconstitutive exons for gRNA targeting. The gRNAs in the library cantarget constitutive exons near the 5′ end of each gene. Optionally, thefirst and last exons of each gene can be excluded as potential targets.Optionally, any exon containing an alternative splicing site can beexcluded as potential targets. Optionally, the two earliest exonsmeeting the above criteria are selected as potential targets.Optionally, exons 2 and 3 are selected as potential targets (e.g., if noconstitutive exons are identified). In addition, the gRNAs in thelibrary can be selected and designed to minimize off-target effects.

In a specific example, the genome-wide CRISPRn gRNA library or librariescomprise sgRNAs targeting 5′ constitutive exons of >18,000 genes in thehuman genome with an average coverage of −6 sgRNAs per gene, with eachtarget site was selected to minimize off-target modification.

The CRISPRa gRNAs can be designed to target sequences adjacent to thetranscription start site of a gene. For example, the target sequence canbe within 1000, 900, 800, 700, 600, 500, 400, 300, 200, 190, 180, 170,160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 40, 30, 20, 10, 5, or1 base pair of the transcription start site. For example, each gRNA inthe CRISPRa library can target a sequence within 200 bp upstream of atranscription start site. Optionally, the target sequence is within theregion 200 base pairs upstream of the transcription start site and 1base pair downstream of the transcription start site (−200 to +1).

The gRNAs in the genome-wide library can be in any form. For example,the gRNA library can be packaged in a viral vector, such as retroviralvectors, lentiviral vectors, or adenoviral vectors. In a specificexample, the gRNA library is packaged in lentiviral vectors. The vectorscan further comprise reporter genes or selection markers to facilitateselection of cells that receive the vectors. Examples of such reportergenes and selection markers are disclosed elsewhere herein. As oneexample, the selection marker can be one that imparts resistance to adrug, such as neomycin phosphotransferase, hygromycin Bphosphotransferase, puromycin-N-acetyltransferase, and blasticidin Sdeaminase. Another exemplary selection marker is bleomycin resistanceprotein, encoded by the Sh ble gene (Streptoalloteichus hindustanusbleomycin gene), which confers zeocin (phleomycin D1) resistance. Forexample, cells can be selected with a drug (e.g., puromycin) so thatonly cells transduced with a guide RNA construct are preserved for beingused to carry out screening.

A. Guide RNAs

A “guide RNA” or “gRNA” is an RNA molecule that binds to a Cas protein(e.g., Cas9 protein) and targets the Cas protein to a specific locationwithin a target DNA. Guide RNAs can comprise two segments: a“DNA-targeting segment” and a “protein-binding segment.” “Segment”includes a section or region of a molecule, such as a contiguous stretchof nucleotides in an RNA. Some gRNAs, such as those for Cas9, cancomprise two separate RNA molecules: an “activator-RNA” (e.g., tracrRNA)and a “targeter-RNA” (e.g., CRISPR RNA or crRNA). Other gRNAs are asingle RNA molecule (single RNA polynucleotide), which can also becalled a “single-molecule gRNA,” a “single-guide RNA,” or an “sgRNA.”See, e.g., WO 2013/176772, WO 2014/065596, WO 2014/089290, WO2014/093622, WO 2014/099750, WO 2013/142578, and WO 2014/131833, each ofwhich is herein incorporated by reference in its entirety for allpurposes. For Cas9, for example, a single-guide RNA can comprise a crRNAfused to a tracrRNA (e.g., via a linker). For Cpf1, for example, only acrRNA is needed to achieve binding to a target sequence. The terms“guide RNA” and “gRNA” include both double-molecule (i.e., modular)gRNAs and single-molecule gRNAs.

An exemplary two-molecule gRNA comprises a crRNA-like (“CRISPR RNA” or“targeter-RNA” or “crRNA” or “crRNA repeat”) molecule and acorresponding tracrRNA-like (“trans-acting CRISPR RNA” or“activator-RNA” or “tracrRNA”) molecule. A crRNA comprises both theDNA-targeting segment (single-stranded) of the gRNA and a stretch ofnucleotides that forms one half of the dsRNA duplex of theprotein-binding segment of the gRNA. An example of a crRNA tail, locateddownstream (3′) of the DNA-targeting segment, comprises, consistsessentially of, or consists of GUUUUAGAGCUAUGCU (SEQ ID NO: 23). Any ofthe DNA-targeting segments disclosed herein can be joined to the 5′ endof SEQ ID NO: 23 to form a crRNA.

A corresponding tracrRNA (activator-RNA) comprises a stretch ofnucleotides that forms the other half of the dsRNA duplex of theprotein-binding segment of the gRNA. A stretch of nucleotides of a crRNAare complementary to and hybridize with a stretch of nucleotides of atracrRNA to form the dsRNA duplex of the protein-binding domain of thegRNA. As such, each crRNA can be said to have a corresponding tracrRNA.An example of a tracrRNA sequence comprises, consists essentially of, orconsists of AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU (SEQ ID NO: 24). Other examples of tracrRNA sequencescomprise, consist essentially of, or consist of any one of

(SEQ ID NO: 28) AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU, or (SEQ ID NO: 29)GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC

In systems in which both a crRNA and a tracrRNA are needed, the crRNAand the corresponding tracrRNA hybridize to form a gRNA. In systems inwhich only a crRNA is needed, the crRNA can be the gRNA. The crRNAadditionally provides the single-stranded DNA-targeting segment thathybridizes to the complementary strand of a target DNA. If used formodification within a cell, the exact sequence of a given crRNA ortracrRNA molecule can be designed to be specific to the species in whichthe RNA molecules will be used. See, e.g., Mali et al. (2013) Science339:823-826; Jinek et al. (2012) Science 337:816-821; Hwang et al.(2013) Nat. Biotechnol. 31:227-229; Jiang et al. (2013) Nat. Biotechnol.31:233-239; and Cong et al. (2013) Science 339:819-823, each of which isherein incorporated by reference in its entirety for all purposes.

The DNA-targeting segment (crRNA) of a given gRNA comprises a nucleotidesequence that is complementary to a sequence on the complementary strandof the target DNA, as described in more detail below. The DNA-targetingsegment of a gRNA interacts with the target DNA in a sequence-specificmanner via hybridization (i.e., base pairing). As such, the nucleotidesequence of the DNA-targeting segment may vary and determines thelocation within the target DNA with which the gRNA and the target DNAwill interact. The DNA-targeting segment of a subject gRNA can bemodified to hybridize to any desired sequence within a target DNA.Naturally occurring crRNAs differ depending on the CRISPR/Cas system andorganism but often contain a targeting segment of between 21 to 72nucleotides length, flanked by two direct repeats (DR) of a length ofbetween 21 to 46 nucleotides (see, e.g., WO 2014/131833, hereinincorporated by reference in its entirety for all purposes). In the caseof S. pyogenes, the DRs are 36 nucleotides long and the targetingsegment is 30 nucleotides long. The 3′ located DR is complementary toand hybridizes with the corresponding tracrRNA, which in turn binds tothe Cas protein.

The DNA-targeting segment can have, for example, a length of at leastabout 12, 15, 17, 18, 19, 20, 25, 30, 35, or 40 nucleotides. SuchDNA-targeting segments can have, for example, a length from about 12 toabout 100, from about 12 to about 80, from about 12 to about from about12 to about 40, from about 12 to about 30, from about 12 to about 25, orfrom about 12 to about 20 nucleotides. For example, the DNA targetingsegment can be from about to about 25 nucleotides (e.g., from about 17to about 20 nucleotides, or about 17, 18, 19, or 20 nucleotides). See,e.g., US 2016/0024523, herein incorporated by reference in its entiretyfor all purposes. For Cas9 from S. pyogenes, a typical DNA-targetingsegment is between 16 and 20 nucleotides in length or between 17 and 20nucleotides in length. For Cas9 from S. aureus, a typical DNA-targetingsegment is between 21 and 23 nucleotides in length. For Cpf1, a typicalDNA-targeting segment is at least 16 nucleotides in length or at least18 nucleotides in length.

TracrRNAs can be in any form (e.g., full-length tracrRNAs or activepartial tracrRNAs) and of varying lengths. They can include primarytranscripts or processed forms. For example, tracrRNAs (as part of asingle-guide RNA or as a separate molecule as part of a two-moleculegRNA) may comprise, consist essentially of, or consist of all or aportion of a wild type tracrRNA sequence (e.g., about or more than about20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild typetracrRNA sequence). Examples of wild type tracrRNA sequences from S.pyogenes include 171-nucleotide, 89-nucleotide, 75-nucleotide, and65-nucleotide versions. See, e.g., Deltcheva et al. (2011) Nature471:602-607; WO 2014/093661, each of which is herein incorporated byreference in its entirety for all purposes. Examples of tracrRNAs withinsingle-guide RNAs (sgRNAs) include the tracrRNA segments found within+48, +54, +67, and +85 versions of sgRNAs, where “+n” indicates that upto the +n nucleotide of wild type tracrRNA is included in the sgRNA. SeeU.S. Pat. No. 8,697,359, herein incorporated by reference in itsentirety for all purposes.

The percent complementarity between the DNA-targeting segment of theguide RNA and the complementary strand of the target DNA can be at least60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 98%, atleast 99%, or 100%). The percent complementarity between theDNA-targeting segment and the complementary strand of the target DNA canbe at least 60% over about 20 contiguous nucleotides. As an example, thepercent complementarity between the DNA-targeting segment and thecomplementary strand of the target DNA can be 100% over the 14contiguous nucleotides at the 5′ end of the complementary strand of thetarget DNA and as low as 0% over the remainder. In such a case, theDNA-targeting segment can be considered to be 14 nucleotides in length.As another example, the percent complementarity between theDNA-targeting segment and the complementary strand of the target DNA canbe 100% over the seven contiguous nucleotides at the 5′ end of thecomplementary strand of the target DNA and as low as 0% over theremainder. In such a case, the DNA-targeting segment can be consideredto be 7 nucleotides in length. In some guide RNAs, at least 17nucleotides within the DNA-targeting segment are complementary to thecomplementary strand of the target DNA. For example, the DNA-targetingsegment can be 20 nucleotides in length and can comprise 1, 2, or 3mismatches with the complementary strand of the target DNA. In oneexample, the mismatches are not adjacent to the region of thecomplementary strand corresponding to the protospacer adjacent motif(PAM) sequence (i.e., the reverse complement of the PAM sequence) (e.g.,the mismatches are in the 5′ end of the DNA-targeting segment of theguide RNA, or the mismatches are at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, or 19 base pairs away from the region ofthe complementary strand corresponding to the PAM sequence).

The protein-binding segment of a gRNA can comprise two stretches ofnucleotides that are complementary to one another. The complementarynucleotides of the protein-binding segment hybridize to form adouble-stranded RNA duplex (dsRNA). The protein-binding segment of asubject gRNA interacts with a Cas protein, and the gRNA directs thebound Cas protein to a specific nucleotide sequence within target DNAvia the DNA-targeting segment.

Single-guide RNAs can comprise a DNA-targeting segment and a scaffoldsequence (i.e., the protein-binding or Cas-binding sequence of the guideRNA). For example, such guide RNAs can have a 5′ DNA-targeting segmentjoined to a 3′ scaffold sequence. Exemplary scaffold sequences comprise,consist essentially of, or consist of:

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (version 1; SEQ ID NO: 17);GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (version 2; SEQ ID NO: 18);GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (version 3; SEQ ID NO: 19); andGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (version 4; SEQ ID NO: 20).Other exemplary scaffold sequences comprise, consist essentially of, orconsist of:

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU (version 5; SEQ ID NO: 30);GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (version 6; SEQ ID NO: 31); orGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU (version 7; SEQ ID NO: 32).Guide RNAs targeting any of the guide RNA target sequences disclosedherein can include, for example, a DNA-targeting segment on the 5′ endof the guide RNA fused to any of the exemplary guide RNA scaffoldsequences on the 3′ end of the guide RNA. That is, any of theDNA-targeting segments disclosed herein can be joined to the 5′ end ofany one of the above scaffold sequences to form a single guide RNA(chimeric guide RNA).

Guide RNAs can include modifications or sequences that provide foradditional desirable features (e.g., modified or regulated stability;subcellular targeting; tracking with a fluorescent label; a binding sitefor a protein or protein complex; and the like). Examples of suchmodifications include, for example, a 5′ cap (e.g., a 7-methylguanylatecap (m7G)); a 3′ polyadenylated tail (i.e., a 3′ poly(A) tail); ariboswitch sequence (e.g., to allow for regulated stability and/orregulated accessibility by proteins and/or protein complexes); astability control sequence; a sequence that forms a dsRNA duplex (i.e.,a hairpin); a modification or sequence that targets the RNA to asubcellular location (e.g., nucleus, mitochondria, chloroplasts, and thelike); a modification or sequence that provides for tracking (e.g.,direct conjugation to a fluorescent molecule, conjugation to a moietythat facilitates fluorescent detection, a sequence that allows forfluorescent detection, and so forth); a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like); and combinations thereof. Otherexamples of modifications include engineered stem loop duplexstructures, engineered bulge regions, engineered hairpins 3′ of the stemloop duplex structure, or any combination thereof. See, e.g., US2015/0376586, herein incorporated by reference in its entirety for allpurposes. A bulge can be an unpaired region of nucleotides within theduplex made up of the crRNA-like region and the minimum tracrRNA-likeregion. A bulge can comprise, on one side of the duplex, an unpaired5′-XXXY-3′ where X is any purine and Y can be a nucleotide that can forma wobble pair with a nucleotide on the opposite strand, and an unpairednucleotide region on the other side of the duplex.

In some cases, a transcriptional activation system can be usedcomprising a dCas9-VP64 fusion protein paired with MS2-p65-HSF1. GuideRNAs in such systems can be designed with aptamer sequences appended tosgRNA tetraloop and stem-loop 2 designed to bind dimerized MS2bacteriophage coat proteins. See, e.g., Konermann et al. (2015) Nature517(7536):583-588, herein incorporated by reference in its entirety forall purposes.

Unmodified nucleic acids can be prone to degradation. Exogenous nucleicacids can also induce an innate immune response. Modifications can helpintroduce stability and reduce immunogenicity. Guide RNAs can comprisemodified nucleosides and modified nucleotides including, for example,one or more of the following: (1) alteration or replacement of one orboth of the non-linking phosphate oxygens and/or of one or more of thelinking phosphate oxygens in the phosphodiester backbone linkage; (2)alteration or replacement of a constituent of the ribose sugar such asalteration or replacement of the 2′ hydroxyl on the ribose sugar; (3)replacement of the phosphate moiety with dephospho linkers; (4)modification or replacement of a naturally occurring nucleobase; (5)replacement or modification of the ribose-phosphate backbone; (6)modification of the 3′ end or 5′ end of the oligonucleotide (e.g.,removal, modification or replacement of a terminal phosphate group orconjugation of a moiety); and (7) modification of the sugar. Otherpossible guide RNA modifications include modifications of or replacementof uracils or poly-uracil tracts. See, e.g., WO 2015/048577 and US2016/0237455, each of which is herein incorporated by reference in itsentirety for all purposes. Similar modifications can be made toCas-encoding nucleic acids, such as Cas mRNAs. For example, Cas mRNAscan be modified by depletion of uridine using synonymous codons.

As one example, nucleotides at the 5′ or 3′ end of a guide RNA caninclude phosphorothioate linkages (e.g., the bases can have a modifiedphosphate group that is a phosphorothioate group). For example, a guideRNA can include phosphorothioate linkages between the 2, 3, or 4terminal nucleotides at the 5′ or 3′ end of the guide RNA. As anotherexample, nucleotides at the 5′ and/or 3′ end of a guide RNA can have2′-O-methyl modifications. For example, a guide RNA can include2′-O-methyl modifications at the 2, 3, or 4 terminal nucleotides at the5′ and/or 3′ end of the guide RNA (e.g., the 5′ end). See, e.g., WO2017/173054 A1 and Finn et al. (2018) Cell Rep. 22(9):2227-2235, each ofwhich is herein incorporated by reference in its entirety for allpurposes. Other possible modifications are described in more detailelsewhere herein. In a specific example, a guide RNA includes2′-O-methyl analogs and 3′ phosphorothioate internucleotide linkages atthe first three 5′ and 3′ terminal RNA residues. Such chemicalmodifications can, for example, provide greater stability and protectionfrom exonucleases to guide RNAs, allowing them to persist within cellsfor longer than unmodified guide RNAs. Such chemical modifications canalso, for example, protect against innate intracellular immune responsesthat can actively degrade RNA or trigger immune cascades that lead tocell death.

In some guide RNAs (e.g., single guide RNAs), at least one loop (e.g.,two loops) of the guide RNA is modified by insertion of a distinct RNAsequence that binds to one or more adaptors (i.e., adaptor proteins ordomains). Such adaptor proteins can be used to further recruit one ormore heterologous functional domains, such as transcriptional activationdomains (e.g., for use in CRISPRa screening in the SAM/tau biosensorcells). Examples of fusion proteins comprising such adaptor proteins(i.e., chimeric adaptor proteins) are disclosed elsewhere herein. Forexample, an MS2-binding loop ggccAACAUGAGGAUCACCCAUGUCUGCAGggcc (SEQ IDNO: 33) may replace nucleotides +13 to +16 and nucleotides +53 to +56 ofthe sgRNA scaffold (backbone) set forth in SEQ ID NO: 17 or SEQ ID NO:19 (or SEQ ID NO: 30 or 31) or the sgRNA backbone for the S. pyogenesCRISPR/Cas9 system described in WO 2016/049258 and Konermann et al.(2015) Nature 517(7536):583-588, each of which is herein incorporated byreference in its entirety for all purposes. See also US 2019-0284572 andWO 2019/183123, each of which is herein incorporated by reference in itsentirety for all purposes. The guide RNA numbering used herein refers tothe nucleotide numbering in the guide RNA scaffold sequence (i.e., thesequence downstream of the DNA-targeting segment of the guide RNA). Forexample, the first nucleotide of the guide RNA scaffold is +1, thesecond nucleotide of the scaffold is +2, and so forth. Residuescorresponding with nucleotides +13 to +16 in SEQ ID NO: 17 or SEQ ID NO:19 (or SEQ ID NO: 30 or 31) are the loop sequence in the region spanningnucleotides +9 to +21 in SEQ ID NO: 17 or SEQ ID NO: 19 (or SEQ ID NO:30 or 31), a region referred to herein as the tetraloop. Residuescorresponding with nucleotides +53 to +56 in SEQ ID NO: 17 or SEQ ID NO:19 (or SEQ ID NO: 30 or 31) are the loop sequence in the region spanningnucleotides +48 to +61 in SEQ ID NO: 17 or SEQ ID NO: 19 (or SEQ ID NO:30 or 31), a region referred to herein as the stem loop 2. Other stemloop sequences in in SEQ ID NO: 17 or SEQ ID NO: 19 (or SEQ ID NO: 30 or31) comprise stem loop 1 (nucleotides +33 to +41) and stem loop 3(nucleotides +63 to +75). The resulting structure is an sgRNA scaffoldin which each of the tetraloop and stem loop 2 sequences have beenreplaced by an MS2 binding loop. The tetraloop and stem loop 2 protrudefrom the Cas9 protein in such a way that adding an MS2-binding loopshould not interfere with any Cas9 residues. Additionally, the proximityof the tetraloop and stem loop 2 sites to the DNA indicates thatlocalization to these locations could result in a high degree ofinteraction between the DNA and any recruited protein, such as atranscriptional activator. Thus, in some sgRNAs, nucleotidescorresponding to +13 to +16 and/or nucleotides corresponding to +53 to+56 of the guide RNA scaffold set forth in SEQ ID NO: 17 or SEQ ID NO:19 (or SEQ ID NO: 30 or 31) or corresponding residues when optimallyaligned with any of these scaffold/backbones are replaced by thedistinct RNA sequences capable of binding to one or more adaptorproteins or domains. Alternatively or additionally, adaptor-bindingsequences can be added to the 5′ end or the 3′ end of a guide RNA. Anexemplary guide RNA scaffold comprising MS2-binding loops in thetetraloop and stem loop 2 regions can comprise, consist essentially of,or consist of the sequence set forth in SEQ ID NO: 34. An exemplarygeneric single guide RNA comprising MS2-binding loops in the tetraloopand stem loop 2 regions can comprise, consist essentially of, or consistof the sequence set forth in SEQ ID NO: 35.

Guide RNAs can be provided in any form. For example, the gRNA can beprovided in the form of RNA, either as two molecules (separate crRNA andtracrRNA) or as one molecule (sgRNA), and optionally in the form of acomplex with a Cas protein. The gRNA can also be provided in the form ofDNA encoding the gRNA. The DNA encoding the gRNA can encode a single RNAmolecule (sgRNA) or separate RNA molecules (e.g., separate crRNA andtracrRNA). In the latter case, the DNA encoding the gRNA can be providedas one DNA molecule or as separate DNA molecules encoding the crRNA andtracrRNA, respectively.

When a gRNA is provided in the form of DNA, the gRNA can be transiently,conditionally, or constitutively expressed in the cell. DNAs encodinggRNAs can be stably integrated into the genome of the cell and operablylinked to a promoter active in the cell. Alternatively, DNAs encodinggRNAs can be operably linked to a promoter in an expression construct.For example, the DNA encoding the gRNA can be in a vector comprising aheterologous nucleic acid, such as a nucleic acid encoding a Casprotein. Alternatively, it can be in a vector or a plasmid that isseparate from the vector comprising the nucleic acid encoding the Casprotein. Promoters that can be used in such expression constructsinclude promoters active, for example, in one or more of a eukaryoticcell, a human cell, a non-human cell, a mammalian cell, a non-humanmammalian cell, a rodent cell, a mouse cell, a rat cell, a pluripotentcell, an embryonic stem (ES) cell, an adult stem cell, a developmentallyrestricted progenitor cell, an induced pluripotent stem (iPS) cell, or aone-cell stage embryo. Such promoters can be, for example, conditionalpromoters, inducible promoters, constitutive promoters, ortissue-specific promoters. Such promoters can also be, for example,bidirectional promoters. Specific examples of suitable promoters includean RNA polymerase III promoter, such as a human U6 promoter, a rat U6polymerase III promoter, or a mouse U6 polymerase III promoter.

Alternatively, gRNAs can be prepared by various other methods. Forexample, gRNAs can be prepared by in vitro transcription using, forexample, T7 RNA polymerase (see, e.g., WO 2014/089290 and WO2014/065596, each of which is herein incorporated by reference in itsentirety for all purposes). Guide RNAs can also be a syntheticallyproduced molecule prepared by chemical synthesis. For example, a guideRNA can be chemically synthesized to include 2′-O-methyl analogs and 3′phosphorothioate internucleotide linkages at the first three 5′ and 3′terminal RNA residues.

Guide RNAs (or nucleic acids encoding guide RNAs) can be in compositionscomprising one or more guide RNAs (e.g., 1, 2, 3, 4, or more guide RNAs)and a carrier increasing the stability of the guide RNA (e.g.,prolonging the period under given conditions of storage (e.g., −20° C.,4° C., or ambient temperature) for which degradation products remainbelow a threshold, such below 0.5% by weight of the starting nucleicacid or protein; or increasing the stability in vivo). Non-limitingexamples of such carriers include poly(lactic acid) (PLA) microspheres,poly(D,L-lactic-coglycolic-acid) (PLGA) microspheres, liposomes,micelles, inverse micelles, lipid cochleates, and lipid microtubules.Such compositions can further comprise a Cas protein, such as a Cas9protein, or a nucleic acid encoding a Cas protein.

B. Guide RNA Target Sequences

Target DNAs for guide RNAs include nucleic acid sequences present in aDNA to which a DNA-targeting segment of a gRNA will bind, providedsufficient conditions for binding exist. Suitable DNA/RNA bindingconditions include physiological conditions normally present in a cell.Other suitable DNA/RNA binding conditions (e.g., conditions in acell-free system) are known in the art (see, e.g., Molecular Cloning: ALaboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press2001), herein incorporated by reference in its entirety for allpurposes). The strand of the target DNA that is complementary to andhybridizes with the gRNA can be called the “complementary strand,” andthe strand of the target DNA that is complementary to the “complementarystrand” (and is therefore not complementary to the Cas protein or gRNA)can be called “noncomplementary strand” or “template strand.”

The target DNA includes both the sequence on the complementary strand towhich the guide RNA hybridizes and the corresponding sequence on thenon-complementary strand (e.g., adjacent to the protospacer adjacentmotif (PAM)). The term “guide RNA target sequence” as used herein refersspecifically to the sequence on the non-complementary strandcorresponding to (i.e., the reverse complement of) the sequence to whichthe guide RNA hybridizes on the complementary strand. That is, the guideRNA target sequence refers to the sequence on the non-complementarystrand adjacent to the PAM (e.g., upstream or 5′ of the PAM in the caseof Cas9). A guide RNA target sequence is equivalent to the DNA-targetingsegment of a guide RNA, but with thymines instead of uracils. As oneexample, a guide RNA target sequence for an SpCas9 enzyme can refer tothe sequence upstream of the 5′-NGG-3′ PAM on the non-complementarystrand. A guide RNA is designed to have complementarity to thecomplementary strand of a target DNA, where hybridization between theDNA-targeting segment of the guide RNA and the complementary strand ofthe target DNA promotes the formation of a CRISPR complex. Fullcomplementarity is not necessarily required, provided that there issufficient complementarity to cause hybridization and promote formationof a CRISPR complex. If a guide RNA is referred to herein as targeting aguide RNA target sequence, what is meant is that the guide RNAhybridizes to the complementary strand sequence of the target DNA thatis the reverse complement of the guide RNA target sequence on thenon-complementary strand.

A target DNA or guide RNA target sequence can comprise anypolynucleotide, and can be located, for example, in the nucleus orcytoplasm of a cell or within an organelle of a cell, such as amitochondrion or chloroplast. A target DNA or guide RNA target sequencecan be any nucleic acid sequence endogenous or exogenous to a cell. Theguide RNA target sequence can be a sequence coding a gene product (e.g.,a protein) or a non-coding sequence (e.g., a regulatory sequence) or caninclude both.

For CRISPRa and SAM systems, it can be preferable for the targetsequence to be adjacent to the transcription start site of a gene. Forexample, the target sequence can be within 1000, 900, 800, 700, 600,500, 400, 300, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100,80, 70, 60, 50, 40, 30, 20, 10, 5, or 1 base pair of the transcriptionstart site. Optionally, the target sequence is within the region 200base pairs upstream of the transcription start site and 1 base pairdownstream of the transcription start site (−200 to +1).

Site-specific binding and cleavage of a target DNA by a Cas protein canoccur at locations determined by both (i) base-pairing complementaritybetween the guide RNA and the complementary strand of the target DNA and(ii) a short motif, called the protospacer adjacent motif (PAM), in thenon-complementary strand of the target DNA. The PAM can flank the guideRNA target sequence. Optionally, the guide RNA target sequence can beflanked on the 3′ end by the PAM (e.g., for Cas9). Alternatively, theguide RNA target sequence can be flanked on the 5′ end by the PAM (e.g.,for Cpf1). For example, the cleavage site of Cas proteins can be about 1to about 10 or about 2 to about 5 base pairs (e.g., 3 base pairs)upstream or downstream of the PAM sequence (e.g., within the guide RNAtarget sequence). In the case of SpCas9, the PAM sequence (i.e., on thenon-complementary strand) can be 5′-NiGG-3′, where N₁ is any DNAnucleotide, and where the PAM is immediately 3′ of the guide RNA targetsequence on the non-complementary strand of the target DNA. As such, thesequence corresponding to the PAM on the complementary strand (i.e., thereverse complement) would be 5′-CCN2-3′, where N2 is any DNA nucleotideand is immediately 5′ of the sequence to which the DNA-targeting segmentof the guide RNA hybridizes on the complementary strand of the targetDNA. In some such cases, N₁ and N₂ can be complementary and the N₁-N₂base pair can be any base pair (e.g., N₁=C and N₂=G; N₁=G and N₂=C; N₁=Aand N₂=T; or N₁=T, and N₂=A). In the case of Cas9 from S. aureus, thePAM can be NNGRRT or NNGRR, where N can A, G, C, or T, and R can be G orA. In the case of Cas9 from C. jejuni, the PAM can be, for example,NNNNACAC or NNNNRYAC, where N can be A, G, C, or T, and R can be G or A.In some cases (e.g., for FnCpf1), the PAM sequence can be upstream ofthe 5′ end and have the sequence 5′-TTN-3′.

An example of a guide RNA target sequence is a 20-nucleotide DNAsequence immediately preceding an NGG motif recognized by an SpCas9protein. For example, two examples of guide RNA target sequences plusPAMs are GNi9NGG (SEQ ID NO: 25) or N₂₀NGG (SEQ ID NO: 26). See, e.g.,WO 2014/165825, herein incorporated by reference in its entirety for allpurposes. The guanine at the 5′ end can facilitate transcription by RNApolymerase in cells. Other examples of guide RNA target sequences plusPAMs can include two guanine nucleotides at the 5′ end (e.g., GGN₂₀NGG;SEQ ID NO: 27) to facilitate efficient transcription by T7 polymerase invitro. See, e.g., WO 2014/065596, herein incorporated by reference inits entirety for all purposes. Other guide RNA target sequences plusPAMs can have between 4-22 nucleotides in length of SEQ ID NOS: 25-27,including the 5′ G or GG and the 3′ GG or NGG. Yet other guide RNAtarget sequences plus PAMs can have between 14 and 20 nucleotides inlength of SEQ ID NOS: 25-27.

Formation of a CRISPR complex hybridized to a target DNA can result incleavage of one or both strands of the target DNA within or near theregion corresponding to the guide RNA target sequence (i.e., the guideRNA target sequence on the non-complementary strand of the target DNAand the reverse complement on the complementary strand to which theguide RNA hybridizes). For example, the cleavage site can be within theguide RNA target sequence (e.g., at a defined location relative to thePAM sequence). The “cleavage site” includes the position of a target DNAat which a Cas protein produces a single-strand break or a double-strandbreak. The cleavage site can be on only one strand (e.g., when a nickaseis used) or on both strands of a double-stranded DNA. Cleavage sites canbe at the same position on both strands (producing blunt ends; e.g.Cas9)) or can be at different sites on each strand (producing staggeredends (i.e., overhangs); e.g., Cpf1). Staggered ends can be produced, forexample, by using two Cas proteins, each of which produces asingle-strand break at a different cleavage site on a different strand,thereby producing a double-strand break. For example, a first nickasecan create a single-strand break on the first strand of double-strandedDNA (dsDNA), and a second nickase can create a single-strand break onthe second strand of dsDNA such that overhanging sequences are created.In some cases, the guide RNA target sequence or cleavage site of thenickase on the first strand is separated from the guide RNA targetsequence or cleavage site of the nickase on the second strand by atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250,500, or 1,000 base pairs.

IV. Methods of Dropout Screening to Reveal Genetic VulnerabilitiesAssociated with Tau Aggregation

The Cas/tau biosensor cell lines disclosed herein can be used in methodsof dropout screening to reveal genetic vulnerabilities associated withtau aggregation. Such methods can comprise providingaggregation-positive and aggregation-negative populations of Cas/taubiosensor cells as disclosed elsewhere herein, introducing a librarycomprising a plurality of unique guide RNAs into each population, anddetermining abundance (e.g., read counts) of each of the plurality ofunique guide RNAs at a plurality of time points over a time course ineach population of cells.

As one example, a method can comprise providing an aggregation-positivepopulation of cells and an aggregation-negative population of cells,wherein each population of cells comprises a Cas protein, a first taurepeat domain linked to a first reporter, and a second tau repeat domainlinked to a second reporter as disclosed elsewhere herein. In theaggregation-positive population of cells the first tau repeat domainlinked to the first reporter and the second tau repeat domain linked tothe second reporter stably present in an aggregated state, whereas inthe aggregation-negative population of cells the first tau repeat domainlinked to the first reporter and the second tau repeat domain linked tothe second reporter do not stably present in an aggregated state. Themethod can then comprise introducing into each population of cells alibrary comprising a plurality of unique guide RNAs that target aplurality of genes. The plurality of unique guide RNAs form complexeswith the Cas protein, and the Cas protein cleaves the plurality of genesresulting in knockout of gene function. Finally, abundance of each ofthe plurality of unique guide RNAs can be determined at a plurality oftime points over a time course in each population of cells. Depletion ofa guide RNA in the aggregation-positive population of cells but not inthe aggregation-negative population of cells can indicate that the genetargeted by the guide RNA exhibits synthetic lethality with tau proteinaggregates and is a genetic vulnerability associated with tauaggregation or is a candidate genetic vulnerability associated tauaggregation (e.g., for further testing via secondary screens), whereindisruption of the gene targeted by the guide RNA is expected to exhibitsynthetic lethality with tau protein aggregates. Alternatively, a moredramatic depletion pattern of a guide RNA over the time course in theaggregation-positive population of cells relative to theaggregation-negative population of cells can indicate that the genetargeted by the guide RNA exhibits synthetic lethality with tau proteinaggregates and is a genetic vulnerability associated with tauaggregation or is a candidate genetic vulnerability associated tauaggregation (e.g., for further testing via secondary screens), whereindisruption of the gene targeted by the guide RNA is expected to exhibitsynthetic lethality with tau protein aggregates. A more dramaticdepletion pattern means that the guide RNA is depleted at a faster rateover the time course (e.g., a distinguishable depletion pattern using adifferential test on fitted exponential decay rates).

Similarly, the SAM/tau biosensor cell lines disclosed herein can be usedin methods of dropout screening to reveal genetic vulnerabilitiesassociated with tau aggregation. Such methods can comprise providingaggregation-positive and aggregation-negative populations of SAM/taubiosensor cells as disclosed elsewhere herein, introducing a librarycomprising a plurality of unique guide RNAs into each population, anddetermining abundance (e.g., read counts) of each of the plurality ofunique guide RNAs at a plurality of time points over a time course ineach population of cells.

As one example, a method can comprise providing an aggregation-positivepopulation of cells and an aggregation-negative population of cells,wherein each population of cells comprises a chimeric Cas proteincomprising a nuclease-inactive Cas protein fused to one or moretranscriptional activation domains, a chimeric adaptor proteincomprising an adaptor protein fused to one or more transcriptionalactivation domains, a first tau repeat domain linked to a firstreporter, and a second tau repeat domain linked to a second reporter asdisclosed elsewhere herein. In the aggregation-positive population ofcells the first tau repeat domain linked to the first reporter and thesecond tau repeat domain linked to the second reporter stably present inan aggregated state, whereas in the aggregation-negative population ofcells the first tau repeat domain linked to the first reporter and thesecond tau repeat domain linked to the second reporter do not stablypresent in an aggregated state. The method can then comprise introducinginto each population of cells a library comprising a plurality of uniqueguide RNAs that target a plurality of genes. The plurality of uniqueguide RNAs form complexes with the chimeric Cas protein and the chimericadaptor protein, and the complexes activate transcription of theplurality of genes resulting in increased gene expression. Finally,abundance of each of the plurality of unique guide RNAs can bedetermined at a plurality of time points over a time course in eachpopulation of cells. Depletion of a guide RNA in theaggregation-positive population of cells but not in theaggregation-negative population of cells can indicate that the genetargeted by the guide RNA, when transcriptionally activated, exhibitssynthetic lethality with tau protein aggregates and is a geneticvulnerability associated with tau aggregation or is a candidate geneticvulnerability associated tau aggregation (e.g., for further testing viasecondary screens), wherein transcriptional activation of the genetargeted by the guide RNA is expected to exhibit synthetic lethalitywith tau protein aggregates. Alternatively, a more dramatic depletionpattern of a guide RNA over the time course in the aggregation-positivepopulation of cells relative to the aggregation-negative population ofcells can indicate that the gene targeted by the guide RNA, whentranscriptionally activated, exhibits synthetic lethality with tauprotein aggregates and is a genetic vulnerability associated with tauaggregation or is a candidate genetic vulnerability associated tauaggregation (e.g., for further testing via secondary screens), whereintranscriptional activation of the gene targeted by the guide RNA isexpected to exhibit synthetic lethality with tau protein aggregates. Amore dramatic depletion pattern means that the guide RNA is depleted ata faster rate over the time course (e.g., a distinguishable depletionpattern using a differential test on fitted exponential decay rates).

The Cas/tau biosensor cells used in the method can be any of the Cas/taubiosensor cells disclosed elsewhere herein. Likewise, the SAM/taubiosensor cells used in the method can be any of the SAM/tau biosensorcells disclosed elsewhere herein. The first tau repeat domain and thesecond tau repeat domain can be different or can be similar or the same.The tau repeat domain can be any of the tau repeat domains disclosedelsewhere herein. For example, the first tau repeat domain and/or thesecond tau repeat domain can be a wild type tau repeat domain or cancomprise a pro-aggregation mutation, such as a tau P301S mutation. Thefirst tau repeat domain and/or the second tau repeat domain can comprisea tau four-repeat domain. As one specific example, the first tau repeatdomain and/or the second tau repeat domain can comprise, consistessentially of, or consist of SEQ ID NO: 11 or a sequence at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO: 11. In one specific example, the nucleic acid encoding the taurepeat domain can comprise, consist essentially of, or consist of SEQ IDNO: 12 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 12, optionally wherein thenucleic acid encodes a protein comprising, consisting essentially of, orconsisting of SEQ ID NO: 11.

The first tau repeat domain can be linked to the first reporter and thesecond tau repeat domain can be linked to the second reporter by anymeans. For example, the reporter can be fused to the tau repeat domain(e.g., as part of a fusion protein). The reporter proteins can be anypair of reporter proteins that produce a detectable signal when thefirst tau repeat domain linked to the first reporter is aggregated withthe second tau repeat domain linked to the second reporter. As oneexample, the first and second reporters can be a split luciferaseprotein. As another example, the first and second reporter proteins canbe a fluorescence resonance energy transfer (FRET) pair. FRET is aphysical phenomenon in which a donor fluorophore in its excited statenon-radiatively transfers its excitation energy to a neighboringacceptor fluorophore, thereby causing the acceptor to emit itscharacteristic fluorescence. Examples of FRET pairs (donor and acceptorfluorophores) are well known. See, e.g., Bajar et al. (2016) Sensors(Basel) 16(9):1488, herein incorporated by reference in its entirety forall purposes. As one specific example of a FRET pair, the first reportercan be cyan fluorescent protein (CFP) and the second reporter can beyellow fluorescent protein (YFP). As a specific example, the CFP cancomprise, consist essentially of, or consist of SEQ ID NO: 13 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 13. As another specific example, the YFP cancomprise, consist essentially of, or consist of SEQ ID NO: 15 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 15.

For the Cas/tau biosensor cells, the Cas protein can be any Cas proteindisclosed elsewhere herein. As one example, the Cas protein can be aCas9 protein. For example, the Cas9 protein can be a Streptococcuspyogenes Cas9 protein. As one specific example, the Cas protein cancomprise, consist essentially of, or consist of SEQ ID NO: 21 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 21.

One or more or all of the Cas protein, the first tau repeat domainlinked to the first reporter, and the second tau repeat domain linked tothe second reporter can be stably expressed in the population of cells.For example, nucleic acids encoding one or more or all of the Casprotein, the first tau repeat domain linked to the first reporter, andthe second tau repeat domain linked to the second reporter can begenomically integrated in the population of cells. In one specificexample, the nucleic acid encoding the Cas protein can comprise, consistessentially of, or consist of SEQ ID NO: 22 or a sequence at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO: 22, optionally wherein the nucleic acid encodes a proteincomprising, consisting essentially of, or consisting of SEQ ID NO: 21.

For the SAM/tau biosensor cells, the Cas protein can be any Cas proteindisclosed elsewhere herein. As one example, the Cas protein can be aCas9 protein. For example, the Cas9 protein can be a Streptococcuspyogenes Cas9 protein. As one specific example, the chimeric Cas proteincan comprise the nuclease-inactive Cas protein fused to a VP64transcriptional activation domain. For example, the chimeric Cas proteincan comprise from N-terminus to C-terminus: the nuclease-inactive Casprotein; a nuclear localization signal; and the VP64 transcriptionalactivator domain. As one specific example, the adaptor protein can be anMS2 coat protein, and the one or more transcriptional activation domainsin the chimeric adaptor protein can comprise a p65 transcriptionalactivation domain and an HSF1 transcriptional activation domain. Forexample, the chimeric adaptor protein can comprise from N-terminus toC-terminus: the MS2 coat protein; a nuclear localization signal; the p65transcriptional activation domain; and the HSF1 transcriptionalactivation domain. In one specific example, the nucleic acid encodingthe chimeric Cas protein can comprise, consist essentially of, orconsist of SEQ ID NO: 38 or a sequence at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38,optionally wherein the nucleic acid encodes a protein comprising,consisting essentially of, or consisting of SEQ ID NO: 36. In onespecific example, the nucleic acid encoding the chimeric adaptor proteincan comprise, consist essentially of, or consist of SEQ ID NO: 39 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 39, optionally wherein the nucleic acidencodes a protein comprising, consisting essentially of, or consistingof SEQ ID NO: 37.

One or more or all of the chimeric Cas protein, the chimeric adaptorprotein, the first tau repeat domain linked to the first reporter, andthe second tau repeat domain linked to the second reporter can be stablyexpressed in the population of cells. For example, nucleic acidsencoding one or more or all of the chimeric Cas protein, the chimericadaptor protein, the first tau repeat domain linked to the firstreporter, and the second tau repeat domain linked to the second reportercan be genomically integrated in the population of cells.

As disclosed elsewhere herein, the cells can be any type of cells. Forexample, the cells can be eukaryotic cells, mammalian cells, or humancells (e.g., HEK293T cells or neuronal cells).

The plurality of unique guide RNAs can be introduced into thepopulations of cells by any known means. In some methods, the guide RNAsare introduced into the populations of cells by viral transduction, suchas retroviral, adenoviral, or lentiviral transduction. In a specificexample, the guide RNAs can be introduced by lentiviral transduction.Each of the plurality of unique guide RNAs can be in a separate viralvector. The populations of cells can be infected at any multiplicity ofinfection. For example, the multiplicity of infection can be betweenabout 0.1 and about 1.0, between about 0.1 and about 0.9, between about0.1 and about 0.8, between about and about 0.7, between about 0.1 andabout 0.6, between about 0.1 and about 0.5, between about 0.1 and about0.4, or between about 0.1 and about 0.3. Alternatively, the multiplicityof infection can be less than about 1.0, less than about 0.9, less thanabout 0.8, less than about 0.7, less than about 0.6, less than about0.5, less than about 0.4, less than about 0.3, or less than about 0.2.In a specific example, the multiplicity of infection can be less thanabout 0.3.

The guide RNAs can be introduced into the populations of cells togetherwith a selection marker or reporter gene to select for cells that havethe guide RNAs, and the method can further comprise selecting cells thatcomprise the selection marker or reporter gene. Examples of selectionmarkers and reporter genes are provided elsewhere herein. As oneexample, the selection marker can be one that imparts resistance to adrug, such as neomycin phosphotransferase, hygromycin Bphosphotransferase, puromycin-N-acetyltransferase, and blasticidin Sdeaminase. Another exemplary selection marker is bleomycin resistanceprotein, encoded by the Sh ble gene (Streptoalloteichus hindustanusbleomycin gene), which confers zeocin (phleomycin D1) resistance. Forexample, cells can be selected with a drug (e.g., puromycin) so thatonly cells transduced with a guide RNA construct are preserved for beingused to carry out screening. For example, the drug can be puromycin orzeocin (phleomycin D1).

In some methods, the plurality of unique guide RNAs are introduced at aconcentration selected such that a majority of the cells receive onlyone of the unique guide RNAs. For example, if the guide RNAs are beingintroduced by viral transduction, the cells can be infected at a lowmultiplicity of infection to ensure that most cells receive only oneviral construct with high probability. As one specific example, themultiplicity of infection can be less than about 0.3.

The populations of cells into which the plurality of unique guide RNAsis introduced can be any suitable number of cells. For example, thepopulations of cells can comprise greater than about 50, greater thanabout 100, greater than about 200, greater than about 300, greater thanabout 400, greater than about 500, greater than about 600, greater thanabout 700, greater than about 800, greater than about 900, or greaterthan about 1000 cells per unique guide RNA. In a specific example, thepopulations of cells comprise greater than about 300 cells or greaterthan about 500 cells per unique guide RNA.

The plurality of unique guide RNAs can target any number of genes. Forexample, the plurality of unique guide RNAs can target about 50 or moregenes, about 100 or more genes, about 200 or more genes, about 300 ormore genes, about 400 or more genes, about 500 or more genes, about 1000or more genes, about 2000 or more genes, about 3000 or more genes, about4000 or more genes, about 5000 or more genes, about 10000 or more genes,or about 20000 or more genes. In some methods, the guide RNAs can beselected to target genes in a particular signaling pathway. In somemethods, the library of unique guide RNAs is a genome-wide library.

The plurality of unique guide RNAs can target any number of sequenceswithin each individual targeted gene. In some methods, a plurality oftarget sequences are targeted on average in each of the targetedplurality of genes. For example, about 2 to about 10, about 2 to about9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2to about 5, about 2 to about 4, or about 2 to about 3 unique targetsequences can be targeted on average in each of the targeted pluralityof genes. For example, at least about 2, at least about 3, at leastabout 4, at least about 5, or at least about 6 unique target sequencescan be targeted on average in each of the targeted plurality of genes.As a specific example, about 6 target sequences can be targeted onaverage in each of the targeted plurality of genes. As another specificexample, about 3 to about 6 or about 4 to about 6 target sequences aretargeted on average in each of the targeted plurality of genes.

The guide RNAs can target any desired location in the target genes. Insome CRISPRn methods using the Cas/tau biosensor cells, each guide RNAtargets a constitutive exon if possible. In some methods, each guide RNAtargets a 5′ constitutive exon if possible. In some methods, each guideRNA targets a first exon, a second exon, or a third exon (from the 5′end of the gene) if possible. In some CRISPRa methods using the SAM/taubiosensor cells, each guide RNA can target a guide RNA target sequencewithin 200 bp upstream of a transcription start site, if possible. Insome CRISPRa methods using the SAM/tau biosensor cells, wherein eachguide RNA can comprise one or more adaptor-binding elements to which thechimeric adaptor protein can specifically bind. In one example, eachguide RNA comprises two adaptor-binding elements to which the chimericadaptor protein can specifically bind, optionally wherein a firstadaptor-binding element is within a first loop of each of the one ormore guide RNAs, and a second adaptor-binding element is within a secondloop of each of the one or more guide RNAs. For example, theadaptor-binding element can comprise the sequence set forth in SEQ IDNO: 33. In a specific example, each of one or more guide RNAs is asingle guide RNA comprising a CRISPR RNA (crRNA) portion fused to atransactivating CRISPR RNA (tracrRNA) portion, and the first loop is thetetraloop corresponding to residues 13-16 of SEQ ID NO: 17, 19, 30, or31, and the second loop is the stem loop 2 corresponding to residues53-56 of SEQ ID NO: 17, 19, 30, or 31.

Abundance of guide RNAs can be determined by any suitable means. In aspecific example, abundance is determined by next-generation sequencing.Next-generation sequencing refers to non-Sanger-based high-throughputDNA sequencing technologies. For example, determining abundance of aguide RNA can comprise measuring read counts of the guide RNA.

The time course in step (c) can be any suitable length of time. Forexample, the time course can be at least about 1 week, at least about 2weeks, more than about 1 week, or more than about 2 weeks. For example,the time course can be between about 6 days and about 28 days, betweenabout 9 days and about 25 days, between about 12 days and about 22 days,between about 15 days and about 19 days, or about 17 days. Similarly,the time course in step (c) can comprise any suitable number of cellpassages or cell doublings. The time course can comprise about 5 toabout 20, about 6 to about 19, about 7 to about 18, about 8 to about 17,about 9 to about 16, about 10 to about 15, about 11 to about 14, about12 to about 13, or about 12 cell doublings. Alternatively, the timecourse can comprise about 2 to about 8, about 2 to about 7, about 2 toabout 6, about 3 to about 5, about 3 to about 6, about 3 to about 7,about 3 to about 8, or about 4 cell passages. For example, there can beabout 10 to about 15 cell doublings. In a specific example, there can beabout 12 cell doublings between the first collection at day 3 and day 17(i.e., about 4 cell passages). In some methods, to maintain a goodrepresentation of the genome wide gRNA library, the cells are notdiluted too much at each passage. For example, the cells can be passagedabout 1/10, about 1/9, about ⅛, about 1/7, about ⅙, about ⅕, about ¼,about ⅓, or about ½. In a specific example, the cells are passaged atabout ⅕ (e.g., so that ≥2 cell doublings).

The tau biosensor cells described herein are primed for aggregation.That is, they are at a tipping point of aggregation. We have found thattau aggregates cause a strong and consistent change in thetranscriptional profile of tau biosensor cells. We hypothesized thatthese aggregation-positive cells may be selectively vulnerable tocertain genetic insults in a way that aggregation-negative cells arenot. We also hypothesized that any “mutation” in the CRISPRn screeningthat would trigger “specific” cell death in aggregate-containing cellsmay also have a lesser deleterious effect on non-aggregate-containingcells. Likewise, any gene activation in the CRISPRa screening that wouldtrigger “specific” cell death in aggregate-containing cells may alsohave a lesser deleterious effect on non-aggregate-containing cells.Assessment at a plurality of time points allows us to identify this typeof target by identifying RNA depletion profiles (depletion rate orsteepness of gRNA depletion) over time rather than only at the lastpassage as compared to the first passage, in which case this type oftarget would be missed. For example, this type of depletion profile wasobserved with Target Gene 1. We identified this type of depletionprofiles for our lead target, Target Gene 1.

The plurality of time points in step (c) can be any suitable number oftime points. For example, the plurality of time points in step (c) canbe at least about 2 time points, at least about 3 time points, at leastabout 4 time points, at least about 5 time points at least about 6 timepoints, more than about 2 time points, more than about 3 time points,more than about 4 time points, more than about 5 time points, or morethan about 6 time points. For example, there can be between about 3 andabout 9 time points, between about 4 and about 8 time points, betweenabout 5 and about 7 time points, or about 6 time points. As anotherexample, there can be between about 2 and about 6 time points, betweenabout 3 and about 5 time points, or about 4 time points.

Each time point can correspond to passaging the cells. The period oftime between each successive time point can be any suitable time period.For example, there can be at least about 12 hours, at least about 1 day,at least about 2 days, at least about 3 days, more than about 12 hours,more than about 1 day, more than about 2 days, or more than about 3 daysbetween each time point. For example, there can be about 1 day and about6 days, about 2 days to about 5 days, or about 3 days to about 4 daysbetween each successive time point. As a specific example, there can beat least about 2 days or about 3 days to about 4 days between eachsuccessive time point (i.e., between successive passages of the cells).

The first time point can be at any suitable time point after the guideRNAs are introduced into the populations of cells. For example, withCRISPRn screening methods using the Cas/tau biosensor cells, the firsttime point can be after a sufficient amount of time for the guide RNAsto form complexes with the Cas protein, and for the Cas protein tocleave the plurality of genes resulting in knockout of gene function.Likewise, with CRISPRa screening methods using the SAM/tau biosensorcells, the first time point can be after a sufficient amount of time forthe guide RNAs to form complexes with the chimeric Cas protein and thechimeric adaptor protein, and for the complexes to activatetranscription of the plurality of genes resulting in increased geneexpression. For example, the first time point can be at least about 1day, at least about 2 days, at least about 3 days, more than about 1day, more than about 2 days, or more than about 3 days afterintroduction of the guide RNAs into the cells. Alternatively, the firsttime point can be between about 0 days and about 6 days, between about 1day and about 5 days, between about 2 days and about 4 days, or about 3days after introduction of the guide RNAs into the cells.

In one specific example, abundance of guide RNAs is assessed at days 3,6 or 7, 10 or 11, 13 or 14, and 17 following introduction of the guideRNAs on day 0. For example, abundance of guide RNAs can be assessed atdays 3, 7, 10, 14, and 17 following introduction of the guide RNAs onday 0.

Whether a guide RNA is considered depleted over the time course caninvolve different assessments. As one example, a guide RNA can beconsidered depleted if the abundance of the guide RNA at each time pointis less than or equal to the abundance of the guide RNA at the precedingtime point. As another example, a guide RNA can be considered depletedin step (c) if the abundance of the guide RNA at each time point afterthe second time point is less than or equal to the abundance of the timepoint two time points prior (i.e., the time point before the precedingtime point, such as time point 3 compared to time point 1, time point 4compared to time point 2, time point 5 compared to time point 3, and soon). In one specific example, abundance of guide RNAs is assessed atdays 3, 6 or 7, 10 or 11, 13 or 14, and 17 following introduction of theguide RNAs on day 0, and a guide RNA is considered depleted if itsabundance at day 10 or 11 is less than at day 3, its abundance at day 13or 14 is less than at day 6 or 7, and its abundance at day 17 is lessthan at day 10 or 11.

The assessment in the methods disclosed herein can also consider thetrend among all guide RNAs in the library that target a particular gene.For example, a gene can be considered to exhibit synthetic lethalitywith tau protein aggregates if at least about 30%, at least about 33%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, more than about 30%, more than about 33%, more thanabout 35%, more than about 40%, more than about 45%, more than about50%, more than about 60%, more than about 65%, more than about 70%, ormore than about 75% (e.g., at least about 30% or more than about 30%) ofthe guide RNAs in the library that target the gene are depleted in theaggregation-positive population of cells but not in theaggregation-negative population of cells. As some specific non-limitingexamples, a gene can be considered to exhibit synthetic lethality withtau protein aggregates (or is expected to exhibit synthetic lethalitywith tau protein aggregates) in the following situations: (1) if thereis one guide RNA in the library that targets the gene, the one guide RNAis depleted in the aggregation-positive population of cells but not inthe aggregation-negative population of cells; (2) if there are two guideRNAs in the library that target the gene, at least one of the two guideRNAs is depleted in the aggregation-positive population of cells but notin the aggregation-negative population of cells; (3) if there are threeguide RNAs in the library that target the gene, at least one of thethree guide RNAs is depleted in the aggregation-positive population ofcells but not in the aggregation-negative population of cells; (4) ifthere are four guide RNAs in the library that target the gene, at leasttwo of the four guide RNAs is depleted in the aggregation-positivepopulation of cells but not in the aggregation-negative population ofcells; (5) if there are five guide RNAs in the library that target thegene, at least two of the five guide RNAs is depleted in theaggregation-positive population of cells but not in theaggregation-negative population of cells; and (6) if there are six guideRNAs in the library that target the gene, at least three of the sixguide RNAs is depleted in the aggregation-positive population of cellsbut not in the aggregation-negative population of cells.

The assessment in the methods disclosed herein can also consider thetrend of guide RNAs in the library that target a particular gene overmultiple experiments. For example, the method can be repeated at leastabout 2 times, at least about 3 times, at least about 4 times, at leastabout 5 times, at least about 6 times, more than about 1 time, more thanabout 2 times, more than about 3 times, more than about 4 times, morethan about 5 times, or more than about 6 times. For example, a gene canbe considered to exhibit synthetic lethality with tau protein aggregatesif it is selected (i.e., if it is considered to exhibit syntheticlethality with tau protein aggregates) in at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, more than about 30%, more than about 35%,more than about 40%, more than about 45%, more than about 50%, more thanabout 55%, more than about 60%, more than about 65%, more than about70%, or more than about 75% of all experiments. For example, a gene canbe considered to exhibit synthetic lethality with tau protein aggregatesif it is selected (i.e., if it is considered to exhibit syntheticlethality with tau protein aggregates) in more than about 50% or morethan about 60% of all experiments. As a specific example, the methodscan be repeated in three experiments, and the gene can be considered toexhibit synthetic lethality with tau protein aggregates (or can beselected as a candidate for exhibiting synthetic lethality with tauprotein aggregates (e.g., for further testing in secondary screens)) ifit is selected (i.e., if it is considered to exhibit synthetic lethalitywith tau protein aggregates) in at least two out of the threeexperiments.

All patent filings, websites, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise, if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

BRIEF DESCRIPTION OF THE SEQUENCES

The nucleotide and amino acid sequences listed in the accompanyingsequence listing are shown using standard letter abbreviations fornucleotide bases, and three-letter code for amino acids. The nucleotidesequences follow the standard convention of beginning at the 5′ end ofthe sequence and proceeding forward (i.e., from left to right in eachline) to the 3′ end. Only one strand of each nucleotide sequence isshown, but the complementary strand is understood to be included by anyreference to the displayed strand. When a nucleotide sequence encodingan amino acid sequence is provided, it is understood that codondegenerate variants thereof that encode the same amino acid sequence arealso provided. The amino acid sequences follow the standard conventionof beginning at the amino terminus of the sequence and proceedingforward (i.e., from left to right in each line) to the carboxy terminus.

TABLE 2 Description of Sequences. SEQ ID NO Type Description 1 ProteinTau R1 Repeat Domain 2 Protein Tau R2 Repeat Domain 3 Protein Tau R3Repeat Domain 4 Protein Tau R4 Repeat Domain 5 DNA Tau R1 Repeat DomainCoding Sequence 6 DNA Tau R2 Repeat Domain Coding Sequence 7 DNA Tau R3Repeat Domain Coding Sequence 8 DNA Tau R4 Repeat Domain Coding Sequence9 Protein Tau Four-Repeat Domain (R1-R4; amino acids 243-375 offull-length (P10636-8) Tau) 10 DNA Coding Sequence for Tau Four-RepeatDomain (R1-R4; coding sequence for amino acids 243-375 of full-length(P10636-8) Tau) 11 Protein Tau Four-Repeat Domain (R1-R4) with P301SMutation 12 DNA Coding Sequence for Tau Four-Repeat Domain (R1-R4) withP301S Mutation 13 Protein eCFP 14 DNA eCFP Coding Sequence 15 ProteineYFP 16 DNA eYFP Coding Sequence 17 RNA Guide RNA Scaffold V1 18 RNAGuide RNA Scaffold V2 19 RNA Guide RNA Scaffold V3 20 RNA Guide RNAScaffold V4 21 Protein Cas9 22 DNA Cas9 Coding Sequence 23 RNA crRNATail 24 RNA TracrRNA 25 DNA Guide RNA Target Sequence Plus PAM V1 26 DNAGuide RNA Target Sequence Plus PAM V2 27 DNA Guide RNA Target SequencePlus PAM V3 28 RNA TracrRNA v2 29 RNA TracrRNA v3 30 RNA Guide RNAScaffold V5 31 RNA Guide RNA Scaffold V6 32 RNA Guide RNA Scaffold V7 33RNA MS2-binding loop 34 RNA Guide RNA Scaffold with MS2-Binding Loops 35RNA Generic sgRNA with MS2-Binding Loops 36 Protein dCas9-VP64 chimericCas protein 37 Protein MCP-p65-HSF1 chimeric adaptor protein 38 DNA DNAEncoding dCas9-VP64 chimeric Cas protein 39 DNA DNA EncodingMCP-p65-HSF1 chimeric adaptor protein 40 Protein MCP 41 DNA DNA EncodingMCP 42 DNA Lenti dCas9-VP64 43 DNA Lenti MCP-p65-HSF1 Hygro

EXAMPLES Example 1. Development of Genome-Wide CRISPR/Cas9 ScreeningPlatform to Identify Genetic Vulnerabilities Associated with TauAggregation

Abnormal aggregation or fibrillization of proteins is a defining featureof many diseases, notably including a number of neurodegenerativediseases such as Alzheimer's disease (AD), Parkinson's disease (PD),frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS),chronic traumatic encephalopathy (CTE), Creutzfeldt-Jakob disease (CJD),and others. In many of these diseases, the fibrillization of certainproteins into insoluble aggregates is not only a hallmark of disease,but has also been implicated as a causative factor of neurotoxicity.Furthermore, these diseases are characterized by propagation ofaggregate pathology through the central nervous system followingstereotypical patterns, a process which correlates with diseaseprogression. The identification of genes and genetic pathways thatmodify the processes of abnormal protein aggregation, or cell-to-cellpropagation of aggregates, are therefore of great value in betterunderstanding neurodegenerative disease etiology as well as in devisingstrategies for therapeutic intervention.

To identify genes and pathways that exhibit synthetic lethality withdisease-associated protein aggregates, a platform was developed forperforming genome-wide screens with CRISPR nuclease (CRISPRn) sgRNAlibraries. The platform identifies genes that, when disrupted, causecell death specifically in the context of abnormal protein aggregation.The identification of such genes may elucidate the mechanisms ofaggregate-associated neurotoxicity, and genetic pathways that promotedeath of neurons in the context of neurodegenerative disease.

The screen employed a Tau biosensor human cell line consisting ofHEK293T cells stably expressing Tau four-repeat domain, Tau_4RD,comprising the Tau microtubule binding domain (MBD) with the P301Spathogenic mutation, fused to either CFP or YFP. That is, the HEK293Tcell lines contain two transgenes stably expressing disease-associatedprotein variants fused to the fluorescent protein CFP or the fluorescentprotein YFP: Tau^(4RD)-CFP/Tau^(4RD)-YFP (TCY), wherein the Tau repeatdomain (4RD) comprises the P301S pathogenic mutation. See FIG. 1 . Inthese biosensor lines, Tau-CFP/Tau-YFP protein aggregation produces aFRET signal, the result of a transfer of fluorescent energy from donorCFP to acceptor YFP. See FIG. 2 . FRET-positive cells, which contain Tauaggregates, can be sorted and isolated by flow cytometry. At baseline,unstimulated cells express the reporters in a stable, soluble state withminimal FRET signal. Upon stimulation (e.g., liposome transfection ofseed particles), the reporter proteins form aggregates, producing a FRETsignal. Aggregate-containing cells can be isolated by FACS. Stablypropagating aggregate-containing cell lines, Agg[+], can be isolated byclonal serial dilution of Agg[−] cell lines.

Several modifications were made to this Tau biosensor cell line to makeit useful for genetic screening. First, these Tau biosensor cells weremodified by introducing a Cas9-expressing transgene via a lentiviralvector. Clonal transgenic cell lines expressing Cas9 were selected withblasticidin and isolated by clonal serial dilution to obtainsingle-cell-derived clones. Clones were evaluated for level of Cas9expression by qRT-PCR (FIG. 3A) and for DNA cleavage activity by digitalPCR (FIG. 3B). Relative Cas9 expression levels are also shown in Table3.

TABLE 3 Relative Cas9 Expression Levels. Cas9D B2m Cas9D- Cas9D Ct AVGAVG B2m Clone Name rep1 rep2 rep3 rep4 Ct Ct delta Ct 3B5-B1 26.22 26.3126.36 26.45 26.33 22.01  4.33 3B5-G2 23.68 23.85 24.39 23.61 23.88 21.51 2.38 7B5-B2 23.63 23.60 24.12 23.50 23.71 21.38  2.34 3B3-A2 24.0523.95 24.02 24.47 24.12 21.94  2.19 7B10-C3 22.58 22.71 22.67 23.2022.79 21.19  1.59 3B5-E1 24.12 24.32 24.75 24.05 24.31 22.81  1.503B5-G1 21.16 21.14 21.09 21.43 21.20 21.35 −0.15 7B5-C3 19.98 19.9919.86 19.97 19.95 21.24 −1.29 7B5-A2 18.84 18.74 19.33 18.99 18.97 22.10−3.12 7B5-G1 19.01 18.88 19.61 19.18 19.17 22.33 −3.16

Specifically, Cas9 mutation efficiency was assessed by digital PCR 3 and7 days after transduction of lentiviruses encoding gRNAs against twoselected target genes. Cutting efficiency was limited by Cas9 levels inlower-expressing clones. A clone with an adequate level of Cas9expression was needed to achieve maximum activity. Several derivedclones with lower Cas9 expression were not able to cut target sequencesefficiently, whereas clones with higher expression (including those usedfor screening) were able to generate mutations at target sequences inthe genes PERK and SNCA with approximately 80% efficiency after threedays in culture. Efficient cutting was observed already at 3 days aftergRNA transduction with only marginal improvement after 7 days. Clone7B10-C3 was selected as a high-performing clone to use for subsequentlibrary screens.

Second, sub-clones of these Cas9-expressing Tau-CFP/Tau-YFP (TCY)biosensor cell lines in which Tau protein is stably present in either anon-aggregated (the default state) (Agg[−]) or an aggregated state(Agg[+]) were obtained. To obtain cell lines in which Tau protein isstably and persistently present in an aggregated state, theCas9-expressing cells were treated with recombinant fibrillized Taumixed with lipofectamine reagent in order to “seed” the aggregation ofthe Tau protein transgenically expressed by these cells. See FIG. 5 .The “seeded” cells were then serially diluted to obtain singlecell-derived clones, and these clones were then expanded to identifythose clonal cell lines in which Tau aggregates stably persist in allcells with growth and multiple passages over time.

We then analyzed the Agg[+] and Agg[−] subclones to determine whethercertain genes were perturbed by Tau aggregation. See FIG. 6 .Unsupervised clustering indicated that the Agg[+] subclones and Agg[−]subclones were distinct by aggregation status (clustering procedure: logtransformation of reads per kilobase million (RPKM); distance metric:1−absolute value of Pearson correlation coefficient; hierarchicalclustering). See FIG. 7 . Unsupervised clustering refers to clusteringsamples without any prior knowledge about the samples (e.g., what cellline a sample is from (Agg[+] or Agg[−])) such that the clusterformation among samples is completely driven by the data itself. Theprocedure was as follows: (1) add 1 to the expression level of all genes(unit is RPKM) in each sample; (2) log 2 transform the RPKM+1 values;(3) calculate pair-wise Pearson correlation coefficient among allsamples; (4) calculate similarity between ever pair of samples as1-absolute value of the Pearson correlation coefficient; and (5) applystandard hierarchical clustering algorithm on values from (4). The datashowed that samples from Agg[+] and Agg[−] cell lines form distinctlyseparated clusters solely based on their gene expression profiles,suggesting Agg[+] and Agg[−] cell lines are different at the geneexpression level.

RNA sequencing of Tau biosensor Agg[+] and Agg[−] subclones revealedthat >1500 genes are perturbed by Tau aggregation. See FIG. 8 .Significantly perturbed genes were defined as those having a fold change≥1.5 in either direction and a p value ≤0.01. RNAseq of five Agg[+]sub-clones and five Agg[−] sub-clones demonstrated that the presence ofTau aggregates causes a strong and consistent change in thetranscriptional profile of Agg[+] sub-clones as compared to Agg[−]sub-clones. Thus, Agg[+] sub-clones may be selectively vulnerable tocertain genetic insults in a way that Agg[−] sub-clones are not. Asexplained in Example 2, we probed these vulnerabilities using CRISPRlibraries to perform a dropout screen and identify sgRNAs that causesynthetic lethality when in combination with Tau aggregates.

Example 2. Genome-Wide CRISPR/Cas9 Screening to Identify GeneticVulnerabilities Associated with Tau Aggregation

To probe the vulnerabilities identified in Example 1, a pooledgenome-wide CRISPR nuclease (CRISPRn) screen was performed to revealgenetic vulnerabilities associated with Tau aggregation. Specifically,the Cas9-expressing Tau-CFP/Tau-YFP biosensor cells, either withaggregates (Agg[+]) or without aggregates (Agg[−]), were transduced withtwo human genome-wide CRISPR sgRNA libraries using a lentiviral deliveryapproach to introduce knock-out mutations at each target gene at arepresentation of 500× cells per sgRNA. See FIGS. 4 and 9 . The CRISPRsgRNA libraries target 5′ constitutive exons for functional knock-outwith an average coverage of 3-4 sgRNAs per gene. The sgRNAs weredesigned to avoid off-target effects by avoiding sgRNAs with two orfewer mismatches to off-target genomic sequences. The libraries cover19,050 human genes and 1864 miRNA with 1000 non-targeting controlsgRNAs. The libraries were transduced at a multiplicity of infection(MOI) <0.3 at a coverage of >500 cells per sgRNA. Tau biosensor cellswere grown under puromycin selection to select cells with integrationand expression of a unique sgRNA per cell. Puromycin selection began 24h after transduction at 1 μg/mL.

Samples of (Agg[+]) and (Agg[−]) cells were collected at five timepoints: Days 3, 6-7, 10-11, 13-14, and 17 post-transduction. DNAisolation and PCR amplification of the integrated sgRNA constructsallowed a characterization by next generation sequencing (NGS) of thesgRNA repertoire in each cell line at each time point. The screeningconsisted of 3 replicated experiments. Analysis of the NGS data enabledthe identification of essential genes, consisting of those genes whosetargeting sgRNAs become depleted over time in both the Agg[+] and Agg[−]cell lines. See FIG. 10A. More importantly, this screening methodenabled the identification of synthetic lethal genes, consisting ofgenes whose targeting sgRNAs become depleted over time preferentially inthe Agg[+] as compared to the Agg[−] cell lines. See FIG. 10B.

Depletion profiles were assessed using a newly defined time courseanalysis in which sgRNAs with a consistent pattern of decreasing NGSreads from the earliest time point (Day 3) to the final time point (Day17) were considered to be depleted. See FIG. 11 and Table 4.

TABLE 4 Time Course Strategy for CRISPRn Dropout Screen. Tau Bio-Samples Collected from 3 Replicate Experiments sensor LV Passage PassagePassage Passage Final Lines Library 1 2 3 4 Collection Agg[−] LibraryDay3 Day 6-7 Day 10-11 Day 13-14 Day 17 A Library B Agg[+] Library Day3Day 6-7 Day 10-11 Day 13-14 Day 17 A Library B

This is a novel analytical approach to evaluating sgRNA depletion, ascompared to the more conventional approach of simply comparing the NGSreads of the endpoint cell collection to the first passage. Incomparison, other published genome-wide CRISPRn screens for depletionhave compared CRISPR scores as the ratio of read counts between finalcollection and early passage. In contrast, our approach was to examinethe full time course to identify genes that exhibit a pattern of sgRNAdepletion over time. Time course analysis identified more genes sgRNAsdepleted in the Agg[+] subset than in the Agg[−] subset in all threerepeat experiments.

The first analysis steps included quality control (non-targeted sgRNAs),normalization (by total reads per sample), and flagging“presence”/“absence” per sgRNA per sample with a detection cutoff of 30reads. First, non-targeting sgRNAs were tested. It was confirmed thatthere was no strong or systematic perturbation or depletion of the 1000non-targeting control sgRNAs. Second, experiments were done to show thatthe plasmid sgRNA library and the Day 3 samples were very similar withrespect to read counts of the sgRNAs (no significant sgRNA depletion hadoccurred at Day 3). This was validation for using Day 3 as the referencepoint for the time course analysis.

Depleted genes were then identified via time course analysis. In eachrepeat, depleted sgRNAs with non-increasing temporal patterns (readcount at Day 10≤at Day 3; read count at Day 14≤at Day 6; and read countDay 17≤at Day 10) relative to Day 3 were selected. sgRNAs alreadydepleted at Day 3 (i.e., below the detection limit of 30 at Day 3 andstaying below the detection limit through the rest of the time points)were kept. Fluctuation close to the detection limit was ignored (i.e.,if the read count of a sgRNA was less than or equal to the mean readcounts of sgRNAs that are below the detection limit+2*standard deviationof the read counts of sgRNAs that are below the detection limit, thesgRNA was considered not detected). Genes were selected if they hadsufficient numbers of sgRNAs depleted (1 out of 1 total sgRNAs, 1 out of2 total sgRNAs, 1 out of 3 total sgRNAs, 2 out of 4 total sgRNAs, 2 outof 5 total sgRNAs, or 3 out of 6 total sgRNAs). Genes were retained ifselected in at least 2 out of 3 repeat time course experiments. Next,genes were identified as “essential” if sgRNAs targeting those geneswere depleted in both Agg[+] and Agg[−]. Genes were identified as“synthetic lethal” if sgRNAs targeting those genes were depleted inAgg[+] but not Agg[−] (no depletion in Agg[−] as compared to Agg[+];refinement based on multiple repeats and manual inspection). Byrefinement is meant taking the genes that presented in all threerepeated time-course experiments in the Agg[+] cell line and thenexcluding any gene that presented in at least one experiment in theAgg[−] cell line. Manual inspection involved reviewing the guide RNAsshown to be depleted in one of the Agg[+] experiments, looking for guideRNAs depleted in all Agg[+] experiments but in no Agg[−] experiments orin only one Agg[−] experiment, and confirming that no targets weremissed.

There was no strong or systematic perturbation with the 1000non-targeting control sgRNAs (data not shown). Specifically, for each ofDays 6, 7, 10, 11, 13, 14, and 17 versus Day 3, all experimental repeatswere combined from both Agg[+] and Agg[−]. P-values (adjusted formultiple testing) of changes in read counts were calculated. No gRNA atany time point had a p value <0.05.

Time course analysis revealed 977 genes as “essential” genes with sgRNAshaving temporal patterns of depletion in both Agg[+] and Agg[−]subclones (data not shown). These genes identified as “essential”significantly overlap with genes identified as essential in publicdatasets (see, e.g., Tzelepis et al. (2016) Cell Reports 17:1193-1205and Wang et al. (2015) Science 350(6264):1096-1101, each of which isherein incorporated by references in its entirety for all purposes). SeeFIG. 13 and Table 5.

TABLE 5 Overlap of Essential Genes with Public Datasets. # GenesOverlapping with Tau Biosensor Public Dataset Essential Genes P-ValueEssential in Wang 359 2e−249 Essential in Tzelepis 581 9e−276 Essentialshared by Wang & Tzelepis 331 5e−231

Genes targeted by multiple sgRNAs exhibiting a pattern of depletion inmultiple replicate screens in Agg[+] but not in Agg[−] were selected forfurther validation in a secondary screen as “synthetic lethal” genes.Seventy-one genes were identified as being depleted in all Agg[+]experimental replicates in comparison to Day 3 but not in any Agg[−]experimental replicates. See FIG. 14 . The data for three target genes(Target Gene 1, Target Gene 5, and Target Gene 6) are shown in FIG. 15B.

These 71 genes were identified by analysis of sgRNA depletion in threereplicates, depletion of sgRNAs over multiple time points, and visualinspection of data. Custom GenScript mini-libraries were then generated.Library 1 contained 462 unique targeting sgRNAs (targeting the 71putative synthetic lethal genes with sgRNAs identified as selectivelydepleted in Agg[+] cells and 10 putative essential genes with sgRNAsidentified as depleted in both Agg[+] and Agg[−] cells, each targeted by−6 unique sgRNAs). Library 2 contained 500 unique non-targeting controlsgRNAs that were not altered over the time course experiment. Read countdistribution of the two libraries is shown in FIGS. 16 and 17 ,respectively. This shows that the representation of the two libraries ismaintained after expansion. Pearson's r describe the statisticalcorrelation between the original library and after expansion.

The libraries were then used for a secondary screen of the candidatetargets. See FIG. 18 . Depletion of targeting sgRNAs relative tonon-targeting control sgRNAs was assessed throughout a 17-daytime-course approach in (Agg[+]) and (Agg[−]) clones at a highrepresentation of 10,000× cells per sgRNA. Depletion of sgRNAs to targetgenes relative to control sgRNAs was assessed throughout the time coursein both Agg[+] and Agg[−] clones. First, read counts were normalized bythe sum of the non-targeting sgRNAs. The normalization involvedmultiplying a scalar number to each sample (different samples can havedifferent scalar numbers) such that after the normalization the totalread counts over all non-targeting sgRNAs per sample were the same.After such normalization, non-targeting sgRNAs showed no change asexpected, and many targeting sgRNAs showed time-wise depletion patterns.Using normalization by sum of the non-targeting sgRNAs, only a smallnumber of non-targeting sgRNAs showed perturbation (perturbationcriteria: p value ≤0.01; fold change ≥1.2). The pattern was random, andthe magnitude was small. Besides visually (qualitatively) showing noperturbations in the non-targeting sgRNAs, this showed by statisticalanalysis (p value and fold change) at Day 7 versus Day 3, Day 10 versusDay 3, Day 14 versus Day 3, and Day 17 versus Day 3 that only a smallnumber of non-targeting sgRNAs were perturbed. In addition, which sgRNAsat which time point were perturbed (relative to Day 3) was rather randombecause the heat map did not show a temporal pattern for any of theseperturbed sgRNAs (data not shown). Next, the 10 putative “essential”genes were validated by assessing sgRNA depletion in both Agg[+] andAgg[−] samples. Time course data from replicates for sgRNAs for the tengenes are shown in FIGS. 19A-19C.

Next, patterns of depletion were assessed for the candidate syntheticlethal genes. For each sgRNA, the significance (p value) of the timefactor was calculated (two experiments were used as repeats per timepoint). A standard general linear regression model (GLM) was used: ybeta*t, where y=read count and t=time, was applied to the data (tworepeated experiments at the five time points (Day 3 to Day 17)). Solvingthe GLM resulted in a p-value for beta that defines how significantlybeta is apart from 0 (i.e., the significance of the time factor). Inaddition, each sgRNA was marked if it had a depleting pattern in everyexperiment (Day 10<Day 3; AND Day 14<Day 7, AND Day 17<Day 10). sgRNAswere selected if they had a significant time course p value (≤1e⁻³) anda depleting pattern in every experiment. sgRNAs were then selected ifthey had a distinguishable depletion pattern between Agg[+] and Agg[−](differential test on fitted exponential decay rates). To determinewhether there was a distinguishable depletion pattern, the followingsteps were used: (1) in both Agg[+] and Agg[−] cells, for each sgRNA X,average its read counts over two repeated experiments at each timepoint; (2) fit the five-time-point data from (1) to an exponential decaymodel: Read count=exp(−rate*t) to estimate the rate, resulting in anexpected value of the rate (e.g., rate 1) and its associated standarderror (e.g., standard error 1); and (3) if [rate 1]/[rate 2]≥1.5 or≤1/1.5, and ([rate 1]−[rate 2])/sqrt([standard error 1]²+[standard error2]²)≥1.96 or ≤−1.96, then rate1 and rate2 are statistically different.An example of a distinguishable depletion pattern (i.e., a more dramaticor sharper depletion pattern in Agg[+] cells) is shown in FIG. 20 .Genes were then selected based on the number of sgRNAs selected (2 outof 4 total sgRNAs targeting a gene, or 3 out of 6 total sgRNAs targetinga gene).

Twenty-seven sgRNAs were identified as having sharper depletion patternsin Agg[+] compared to Agg[−]. See FIG. 21 . Of these, four target geneswere selected for further validation (Target Gene 1, Target Gene 2,Target Gene 3, and Target Gene 4) based on the number of sgRNAs selected(2 out of 4 total sgRNAs targeting a gene, or 3 out of 6 total sgRNAstargeting a gene). The validation for Target Genes 1 and 2 is shown inFIG. 22A, and the validation for Target Genes 3 and 4 is shown in FIG.22B. The 10 essential genes were also validated by a secondary screeningmethod (data not shown).

For further validation, the Cas9-expressing Tau-CFP/Tau-YFP biosensorcells, either with aggregates (Agg[+]) or without aggregates (Agg[−]),were transduced with sgRNAs targeting Target Gene 1, Target Gene 2, andTarget Gene 4 using a lentiviral delivery approach to introduceknock-out mutations at each target gene (FIG. 25 ). Following expansionunder selection for 7 days, cell viability and cell death was assessedand quantified by flow cytometry (FIG. 26 ), and decreases in expressionof the target genes was assessed and confirmed (data not shown). ThesgRNAs targeting Target Gene 1, Target Gene 2, and Target Gene 4 causedselective cell death in the Agg[+] cells but not in the Agg[−] cells.

Example 3. Development of a Genome-Wide CRISPR/Cas9 Screening Platformto Identify Genetic Modifiers of Tau Affecting Cell Viability Using aTranscriptional Activation CRISPR/Cas9 Library

To identify genes that when transcriptionally activated exhibitsynthetic lethality with disease-associated protein aggregates, aplatform was developed for performing genome-wide screens withtranscriptional activation (hSAM) CRISPRa sgRNA libraries. The platformidentifies genes that, upon transcriptional activation, cause cell deathspecifically in the context of abnormal protein aggregation. Theidentification of such genes may elucidate the mechanisms ofaggregate-associated neurotoxicity, and genetic pathways that promotedeath of neurons in the context of neurodegenerative disease.

The screen employed a Tau biosensor human cell line consisting ofHEK293T cells stably expressing Tau four-repeat domain, Tau_4RD,comprising the Tau microtubule binding domain (MBD) with the P301Spathogenic mutation, fused to either CFP or YFP. That is, the HEK293Tcell lines contain two transgenes stably expressing disease-associatedprotein variants fused to the fluorescent protein CFP or the fluorescentprotein YFP: Tau^(4RD)-CFP/Tau^(4RD)-YFP (TCY), wherein the Tau repeatdomain (4RD) comprises the P301S pathogenic mutation. See FIG. 1 . Inthese biosensor lines, Tau-CFP/Tau-YFP protein aggregation produces aFRET signal, the result of a transfer of fluorescent energy from donorCFP to acceptor YFP. See FIG. 2 . FRET-positive cells, which contain Tauaggregates, can be sorted and isolated by flow cytometry. At baseline,unstimulated cells express the reporters in a stable, soluble state withminimal FRET signal. Upon stimulation (e.g., liposome transfection ofseed particles), the reporter proteins form aggregates, producing a FRETsignal. Aggregate-containing cells can be isolated by FACS. Stablypropagating aggregate-containing cell lines, Agg[+], can be isolated byclonal serial dilution of Agg[−] cell lines.

Several modifications were made to this Tau biosensor cell line to makeit useful for genetic screening. First, this biosensor cell line wasfurther transgenically modified to express the components of theCRISPR/Cas SAM transcriptional activation system: dCas9-VP64 andMS2-P65-HSF1. Lentiviral dCas9-VP64 and MS2-P65-HSF1 constructs areprovided in SEQ ID NOS: 42 and 43, respectively. Clone DC11 was selectedas a high-performing clone to use for subsequent library screens. Thisclone was validated for its efficacy in activating selected targetgenes.

Second, sub-clones of these SAM-expressing Tau-CFP/Tau-YFP (TCY)biosensor cell lines in which Tau protein is stably present in either anon-aggregated (the default state) (Agg[−]) or an aggregated state(Agg[+]) were obtained. To obtain cell lines in which Tau protein isstably and persistently present in an aggregated state, theSAM-expressing cells were treated with recombinant fibrillized Tau mixedwith lipofectamine reagent in order to “seed” the aggregation of the Tauprotein transgenically expressed by these cells. The “seeded” cells werethen serially diluted to obtain single cell-derived clones, and theseclones were then expanded to identify those clonal cell lines in whichTau aggregates stably persist in all cells with growth and multiplepassages over time. One of these aggregate-positive Agg[+] stableclones, DC11-B6, was selected for expansion and use in screening.

Example 4. Genome-Wide CRISPR/Cas9 Screening Platform to IdentifyGenetic Modifiers of Tau Affecting Cell Viability Using aTranscriptional Activation CRISPR/Cas9 Library

A pooled genome-wide transcriptional activation (hSAM) CRISPRa screenwas performed to reveal genetic modifiers of Tau affecting cellviability using the clonal cell lines developed in Example 3.Specifically, the SAM-expressing (i.e.,dCas9-VP64/MS2-P65-HSF1-expressing) Tau-CFP/Tau-YFP biosensor cells,either with aggregates (Agg[+]) or without aggregates (Agg[−]), weretransduced with a human genome-wide CRISPR hSAM sgRNA library using alentiviral delivery approach to transcriptionally activate each targetgene at a representation of 500× cells per sgRNA. The CRISPR hSAM sgRNAlibrary targets sites within 200 bp upstream of the transcription startsite with an average coverage of 3-4 sgRNAs per gene. The sgRNAs weredesigned to avoid off-target effects by avoiding sgRNAs with two orfewer mismatches to off-target genomic sequences. The library covers18,946 human genes. The library was transduced at a multiplicity ofinfection (MOI) <0.3 at a coverage of >500 cells per sgRNA. Taubiosensor cells were grown under zeocin selection to select cells withintegration and expression of a unique sgRNA per cell.

Samples of (Agg[+]) and (Agg[−]) cells were collected at five timepoints: Days 3, 6, 7, 14, and 17 post-transduction. DNA isolation andPCR amplification of the integrated sgRNA constructs allowed acharacterization by next generation sequencing (NGS) of the sgRNArepertoire in each cell line at each time point. The screening consistedof 4 replicated experiments. Analysis of the NGS data enabled theidentification of genes which, upon transcriptional activation, lead tocell death. sgRNAs that are depleted in both cell lines are likelydisrupting some cell process essential for cell viability, while sgRNAsthat are depleted specifically in the Agg[+] cell line, while not orless-depleted in the Agg[−] cell line, may indicate a synthetic lethaleffect, in which the activation of a specific target gene combines withthe presence of Tau aggregates in the cell to induce cell death. Thesesynthetic lethal genes are of interest as potential modifiers ofTau-associated cell toxicity.

The data analysis mirrored the approach used in Example 2 and included(1) sgRNA profiling in two cell population Agg[+] and Agg[−] throughouta time-course of 17 days and (2) sgRNA profiling in Agg[+] compared toAgg[−] at different time points or at the final time point compared toDay 3. Because the data analysis mirrored the approach used in Example2, not all of the details from Example 2 are repeated here.

As explained in Example 2, this is a novel analytical approach toevaluating sgRNA depletion, as compared to the more conventionalapproach of simply comparing the NGS reads of the endpoint cellcollection to the first passage. In comparison, other publishedgenome-wide CRISPR screens for depletion have compared CRISPR scores asthe ratio of read counts between final collection and early passage. Incontrast, our approach was to examine the full time course to identifygenes that exhibit a pattern of sgRNA depletion over time.

Depleted genes were then identified via time course analysis. In eachrepeat, depleted sgRNAs with non-increasing temporal patterns (readcount at Day 10≤at Day 3; read count at Day 14≤at Day 7; and read countDay 17≤at Day 10) relative to Day 3 were selected. sgRNAs alreadydepleted at Day 3 (i.e., below the detection limit of 30 at Day 3 andstaying below the detection limit through the rest of the time points)were kept. Fluctuation close to the detection limit was ignored (i.e.,if the read count of a sgRNA was less than or equal to the mean readcounts of sgRNAs that are below the detection limit+2*standard deviationof the read counts of sgRNAs that are below the detection limit, thesgRNA was considered not detected). Genes were selected if they hadsufficient numbers of sgRNAs depleted (1 out of 1 total sgRNAs, 1 out of2 total sgRNAs, 1 out of 3 total sgRNAs, 2 out of 4 total sgRNAs, 2 outof 5 total sgRNAs, or 3 out of 6 total sgRNAs). Next, genes wereidentified as “essential” if sgRNAs targeting those genes were depletedin both Agg[+] and Agg[−]. Genes were identified as “synthetic lethal”if sgRNAs targeting those genes were depleted in Agg[+] but not Agg[−](no depletion in Agg[−] as compared to Agg[+]; refinement based onmultiple repeats and manual inspection). By refinement is meant takingthe genes that presented in all three repeated time-course experimentsin the Agg[+] cell line and then excluding any gene that presented in atleast one experiment in the Agg[−] cell line. Manual inspection involvedreviewing the guide RNAs shown to be depleted in one of the Agg[+]experiments, looking for guide RNAs depleted in all Agg[+] experimentsbut in no Agg[−] experiments or in only one Agg[−] experiment, andconfirming that no targets were missed.

Genes targeted by multiple sgRNAs exhibiting a pattern of depletion inmultiple replicate screens in Agg[+] but not in Agg[−] were identifiedas candidate “synthetic lethal” genes. Next, patterns of depletion wereassessed for the candidate synthetic lethal genes. For each sgRNA, thesignificance (p value) of the time factor was calculated (twoexperiments were used as repeats per time point). A standard generallinear regression model (GLM) was used: y beta*t, where y=read count andt=time, was applied to the data (two repeated experiments at the fivetime points (Day 3 to Day 17)). Solving the GLM resulted in a p-valuefor beta that defines how significantly beta is apart from 0 (i.e., thesignificance of the time factor). In addition, each sgRNA was marked ifit had a depleting pattern in every experiment (Day 10<Day 3; AND Day14<Day 7, AND Day 17<Day 10). sgRNAs were selected if they had asignificant time course p value (≤1e⁻³) and a depleting pattern in everyexperiment. sgRNAs were then selected if they had a distinguishabledepletion pattern between Agg[+] and Agg[−] (differential test on fittedexponential decay rates). To determine whether there was adistinguishable depletion pattern, the following steps were used: (1) inboth Agg[+] and Agg[−] cells, for each sgRNA X, average its read countsover two repeated experiments at each time point; (2) fit thefive-time-point data from (1) to an exponential decay model: Readcount=exp(−rate*t) to estimate the rate, resulting in an expected valueof the rate (e.g., rate 1) and its associated standard error (e.g.,standard error 1); and (3) if [rate 1]/[rate 2]≥1.5 or ≤1/1.5, and([rate 1]-[rate 2])/sqrt([standard error 1]²+[standard error 2]²)≥1.96or ≤−1.96, then rate1 and rate2 are statistically different. An exampleof a distinguishable depletion pattern (i.e., a more dramatic or sharperdepletion pattern in Agg[+] cells) is shown in FIG. 20 .

Thirteen sgRNAs representing nine target genes (Target Genes 7-15) wereidentified as having sharper depletion patterns in Agg[+] compared toAgg[−]. See FIG. 23 . The validation for Target Genes 7-15 is shown inFIGS. 24A-24B.

We claim:
 1. A method of screening for genetic vulnerabilitiesassociated with tau aggregation, comprising: (a) providing anaggregation-positive population of cells and an aggregation-negativepopulation of cells, wherein each population of cells comprises achimeric Cas protein comprising a nuclease-inactive Cas protein fused toone or more transcriptional activation domains, a chimeric adaptorprotein comprising an adaptor protein fused to one or moretranscriptional activation domains, a first tau repeat domain linked toa first reporter, and a second tau repeat domain linked to a secondreporter, wherein the cells are mammalian cells, wherein the firstreporter and the second reporter are fluorescent proteins, and whereinthe first reporter and the second reporter are a fluorescence resonanceenergy transfer (FRET) pair, wherein in the aggregation-positivepopulation of cells the first tau repeat domain linked to the firstreporter and the second tau repeat domain linked to the second reporterstably present in an aggregated state, and wherein in theaggregation-negative population of cells the first tau repeat domainlinked to the first reporter and the second tau repeat domain linked tothe second reporter do not stably present in an aggregated state; (b)introducing into each population of cells a library comprising aplurality of unique guide RNAs that target a plurality of genes, whereinthe plurality of unique guide RNAs form complexes with the chimeric Casprotein and the chimeric adaptor protein, and the complexes activatetranscription of the plurality of genes resulting in increased geneexpression; and (c) determining abundance of each of the plurality ofunique guide RNAs at a plurality of time points over a time course ineach population of cells, wherein the plurality of time points comprisesat least three time points, wherein there is more than 1 day betweeneach time point, wherein depletion of a guide RNA in theaggregation-positive population of cells but not in theaggregation-negative population of cells or a more dramatic depletionpattern of a guide RNA over the time course in the aggregation-positivepopulation of cells relative to the aggregation-negative population ofcells indicates that activation of the gene targeted by the guide RNAexhibits synthetic lethality with tau protein aggregates and is agenetic vulnerability associated with tau aggregation.
 2. The method ofclaim 1, wherein the Cas protein is a Cas9 protein.
 3. The method ofclaim 2, wherein the Cas protein is Streptococcus pyogenes Cas9.
 4. Themethod of claim 1, wherein the chimeric Cas protein comprises thenuclease-inactive Cas protein fused to a VP64 transcriptional activationdomain, wherein the chimeric Cas protein comprises from N-terminus toC-terminus: the nuclease-inactive Cas protein; a nuclear localizationsignal; and the VP64 transcriptional activator domain.
 5. The method ofclaim 1, wherein the adaptor protein is an MS2 coat protein, and whereinthe one or more transcriptional activation domains in the chimericadaptor protein comprise a p65 transcriptional activation domain and anHSF1 transcriptional activation domain, wherein the chimeric adaptorprotein comprises from N-terminus to C-terminus: the MS2 coat protein; anuclear localization signal; the p65 transcriptional activation domain;and the HSF1 transcriptional activation domain.
 6. The method of claim1, wherein the chimeric Cas protein comprises SEQ ID NO: 36, or whereinthe chimeric Cas protein is encoded by a coding sequence comprising thesequence set forth in SEQ ID NO:
 38. 7. The method of claim 1, whereinthe chimeric adaptor protein comprises SEQ ID NO: 37, or wherein thechimeric adaptor protein is encoded by a coding sequence comprising thesequence set forth in SEQ ID NO:
 39. 8. The method of claim 1, whereinthe chimeric Cas protein, the chimeric adaptor protein, the first taurepeat domain linked to the first reporter, and the second tau repeatdomain linked to the second reporter are stably expressed in thepopulation of cells and are genomically integrated in the population ofcells.
 9. The method of claim 1, wherein each guide RNA targets a guideRNA target sequence within 200 bp upstream of a transcription startsite.
 10. The method of claim 1, wherein each guide RNA comprises twoadaptor-binding elements to which the chimeric adaptor protein canspecifically bind, wherein a first adaptor-binding element is within afirst loop of each of the guide RNAs, and a second adaptor-bindingelement is within a second loop of each of the guide RNAs, wherein theadaptor-binding element comprises the sequence set forth in SEQ ID NO:33, and wherein each of guide RNAs is a single guide RNA comprising aCRISPR RNA (crRNA) portion fused to a transactivating CRISPR RNA(tracrRNA) portion, and the first loop is the tetraloop corresponding toresidues 13-16 of SEQ ID NO: 17, and the second loop is the stem loop 2corresponding to residues 53-56 of SEQ ID NO:
 17. 11. The method ofclaim 1, wherein the first tau repeat domain and/or the second taurepeat domain is a human tau repeat domain.
 12. The method of claim 1,wherein the first tau repeat domain and/or the second tau repeat domaincomprises a pro-aggregation mutation, wherein the pro-aggregationmutation is a tau P301S mutation.
 13. The method of claim 1, wherein thefirst tau repeat domain and/or the second tau repeat domain comprises atau four-repeat domain.
 14. The method of claim 1, wherein the first taurepeat domain and/or the second tau repeat domain comprises SEQ ID NO:11.
 15. The method of claim 1, wherein the first tau repeat domain andthe second tau repeat domain are the same and each comprises a taufour-repeat domain comprising a tau P301S mutation.
 16. The method ofclaim 1, wherein the first reporter is cyan fluorescent protein (CFP)and the second reporter is yellow fluorescent protein (YFP).
 17. Themethod of claim 1, wherein the cells are human cells.
 18. The method ofclaim 17, wherein the cells are HEK293T cells.
 19. The method of claim1, wherein the plurality of unique guide RNAs are introduced at aconcentration selected such that a majority of the cells receive onlyone of the unique guide RNAs.
 20. The method of claim 1, wherein theplurality of unique guide RNAs target 100 or more genes, 1000 or moregenes, or 10000 or more genes.
 21. The method of claim 1, wherein thelibrary is a genome-wide library.
 22. The method of claim 1, wherein aplurality of target sequences are targeted on average in each of thetargeted plurality of genes.
 23. The method of claim 22, wherein atleast three target sequences are targeted on average in each of thetargeted plurality of genes or wherein about three to about six targetsequences are targeted on average in each of the targeted plurality ofgenes.
 24. The method of claim 1, wherein the plurality of unique guideRNAs are introduced into the populations of cells by lentiviraltransduction, wherein each of the plurality of unique guide RNAs is in aseparate viral vector.
 25. The method of claim 24, wherein thepopulations of cells are infected at a multiplicity of infection of lessthan about 0.3.
 26. The method of claim 1, wherein the plurality ofunique guide RNAs are introduced into the populations of cells togetherwith a selection marker that imparts resistance to a drug, and step (b)further comprises selecting cells that comprise the selection marker.27. The method of claim 1, wherein the populations of cells into whichthe plurality of unique guide RNAs are introduced in step (b) eachcomprise greater than about 500 cells per unique guide RNA.
 28. Themethod of claim 1, wherein the time course in step (c) is more thanabout 1 week.
 29. The method of claim 28, wherein the time course instep (c) is more than about 2 weeks.
 30. The method of claim 1, whereinthe time course in step (c) comprises about 10 to about 15 celldoublings.
 31. The method of claim 1, wherein the plurality of timepoints in step (c) comprises about four time points or about six timepoints.
 32. The method of claim 1, wherein there is more than about 2days between each time point in step (c).
 33. The method of claim 32,wherein there is between about 3 to about 4 days between each time pointin step (c).
 34. The method of claim 1, wherein a gene is considered toexhibit synthetic lethality with tau protein aggregates in step (c) if aguide RNA targeting the gene is depleted in the aggregation-positivepopulation of cells but not in the aggregation-negative population ofcells.
 35. The method of claim 34, wherein a gene is considered toexhibit synthetic lethality with tau protein aggregates in step (c) if aguide RNA targeting the gene has a more dramatic depletion pattern overthe time course in the aggregation-positive population of cells relativeto the aggregation-negative population of cells.
 36. The method of claim1, wherein a guide RNA is considered depleted in step (c) if theabundance of the guide RNA at each time point is less than or equal tothe abundance of the guide RNA at the preceding time point.
 37. Themethod of claim 1, wherein a guide RNA is considered depleted in step(c) if the abundance of the guide RNA at each time point after thesecond time point is less than or equal to the abundance of the timepoint two time points prior.
 38. The method of claim 1, wherein a geneis considered to exhibit synthetic lethality with tau protein aggregatesin step (c) if more than about 30% of the guide RNAs in the library thattarget the gene are depleted in the aggregation-positive population ofcells but not in the aggregation-negative population of cells.
 39. Themethod of claim 38, wherein a gene is considered to exhibit syntheticlethality with tau protein aggregates in step (c) in any one of thefollowing situations: (1) there is one guide RNA in the library thattargets the gene, and the one guide RNA is depleted in theaggregation-positive population of cells but not in theaggregation-negative population of cells; (2) there are two guide RNAsin the library that target the gene, and at least one of the two guideRNAs is depleted in the aggregation-positive population of cells but notin the aggregation-negative population of cells; (3) there are threeguide RNAs in the library that target the gene, and at least one of thethree guide RNAs is depleted in the aggregation-positive population ofcells but not in the aggregation-negative population of cells; (4) thereare four guide RNAs in the library that target the gene, and at leasttwo of the four guide RNAs is depleted in the aggregation-positivepopulation of cells but not in the aggregation-negative population ofcells; (5) there are five guide RNAs in the library that target thegene, and at least two of the five guide RNAs is depleted in theaggregation-positive population of cells but not in theaggregation-negative population of cells; and (6) there are six guideRNAs in the library that target the gene, and at least three of the sixguide RNAs is depleted in the aggregation-positive population of cellsbut not in the aggregation-negative population of cells.
 40. The methodof claim 1, wherein the method is repeated at least three times in atleast three different experiments, and a gene is considered to exhibitsynthetic lethality with tau protein aggregates if it is considered toexhibit synthetic lethality with tau protein aggregates in more thanabout 50% of the at least three different experiments.
 41. The method ofclaim 1, wherein the time course in step (c) is more than about 2 weeks,wherein the plurality of time points in step (c) comprises about sixtime points, wherein there is between about 3 to about 4 days betweeneach time point in step (c), wherein a guide RNA is considered depletedin step (c) if the abundance of the guide RNA at each time point afterthe second time point is less than or equal to the abundance of the timepoint two time points prior, and wherein a gene is considered to exhibitsynthetic lethality with tau protein aggregates in step (c) if more thanabout 30% of the guide RNAs in the library that target the gene aredepleted in the aggregation-positive population of cells but not in theaggregation-negative population of cells.